Methods for improving the health of porcine species by targeted inactivation of cd163

ABSTRACT

The present disclosure relates methods and compositions useful for prevention of porcine reproductive and respiratory syndrome virus (PRRSv) in animals, including animals of the species  Sus scrofa . The present teachings relate to swine wherein at least one allele of a CD163 gene has been inactivated, and to specific methods and nucleic acid sequences used in gene editing to inactivate the CD163 gene. Swine wherein both alleles of the CD163 gene are inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSv). Elite lines comprising homozygous CD163 edited genes retain their superior properties

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S.Non-Provisional application Ser. No. 17/307,369, filed May 4, 2021. Ser.No. 17/307,369 claims priority to U.S. Provisional Application63/020,128 filed on May 5, 2020 and to U.S. Provisional Application63/021,370 filed on May 7, 2020. Each application is hereby incorporatedby reference, each in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII-formatted sequence listing with a file namedRD-12-2020-US2-SEQLST, created on Oct. 27, 2021, and having a size of137,066 bytes is filed concurrently with the specification. The sequencelisting contained in this ASCII-formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to methods for improving the health ofporcine species. In particular, the present disclosure relates tomethods of protecting Sus scrofa animals and elite lines from infectionby porcine reproductive and respiratory syndrome virus (PRRSv) throughtargeted polynucleotide edits of CD163 to prevent infection and generateresistant animals, herds, and cell lines.

BACKGROUND

Viral infections are a major source of morbidity and mortality in thelivestock industry. In particular, Porcine Reproductive and RespiratorySyndrome (PRRS) is a panzootic infectious disease of pigs, causing majoreconomic losses to the world-wide pig industry. PRRS manifests in pigsof all ages but primarily causes late-term abortions and stillbirths insows and respiratory disease in piglets. The causative agent of thedisease is the positive-strand RNA PRRS virus (PRRSv). PRRS is the mosteconomically important disease of domestic swine in North America,Europe and Asia, costing producers in North America more than $600million annually.

Currently, there are no effective treatment programs for acute PRRS. Asa result, when incidence of PRRS is detected on a farm, depopulation,sufficient cleaning/disinfection, and proper disposal of the carcassesmust be used to eliminate the virus. In more extreme cases, whole herddepopulation-repopulation has been documented as an effective method ofeliminating the PRRSv from endemically infected herds; however, thismethod results in significant loss.

Vaccines for PRRSv do exist; however, these vaccines have been unable tocontrol the disease largely due to the genetic diversity within thestructural proteins of the virus. Consequently, prevention of infectionis currently the best control measure. As a prophylactic measure, farmsin a country or zone where PRRSv exists must use stringent controlmeasures, involving an assessment of the health status of replacementgilts and boars, as well as 45-60 days of isolation and acclimatizationfor incoming stock.

In recent years, more attention has been given to the role CD163 mayplay in the occurrence of PRRS. Despite the significant heterogeneity instrains of PRRSv, strains of PRRSv all share a tropism forCD163-positive cells. Although CD163 is a virus receptor, the CD163scavenger receptor is also involved in the adhesion of monocytes toendothelial cells. Functions and a detailed description of CD163 areprovided in Onofre, Gabriela et al., ACTA MEDICA, 2009, 52, 57-61.

CD163 is a 130 kDa type 1 membrane protein considered to be a fusionreceptor for the PRRS virus; it is mapped to chromosome 5 in pigs. Thebasic transcript encodes for a protein of 1076 amino acids. There arefive reported isoforms of CD163; three of the isoforms display differentsplicing forms of their cytoplasmic domains. Generally, however, thegenomic molecule sequence of CD163 comprises 17 exons coding for apeptide signal sequence, nine scavenger receptor cysteine-rich (SRCR)domains, two proline serine threonine (PST) linker domains, acytoplasmic domain, and a short cytoplasmic tail. CD163 has beendescribed as the receptor for PRRSv. Domain 5 (SRCR5) of the protein isthe interaction site for the virus. Exon 7 of CD163 encodes the SRCRdomain 5 (SRCR5) that serves as an interaction site for the PRRSv invitro. Burkard (Burkard, C., PLoS Pathog. 2017, 23, 13, e1006206.)demonstrated that removal of CD163 exon 7 confers PRRSv resistance toporcine macrophages. The guides used in that work (set forth astargeting sequences including the PAM in SEQ ID NOs: 272 and 273),however, may lack sufficient activity and specificity for gene editingas part of a commercial breeding program. Further work by Whitworth andcolleagues included creating a 123 bp deletion in Exon 7 using guides asset forth (including the PAM) in SEQ ID NO: 354 and SEQ ID NO: 211(Whitworth, K. M., Biol. Reprod., 2014, 91, 1-13). Whitworth et al.(Whitworth, K. M., Nature Biotechnology, 2016, 34, 20-22) reported thepreparation of PRRSv resistant pigs by knocking out the function ofCD163.

Genome editing includes altering the genome by deleting, inserting, orsubstituting specific nucleic acid sequences. The alteration can begene- or location-specific. Genome editing can use site-directednucleases, such as Cas proteins and their cognate polynucleotides.

Clustered regularly interspaced short palindromic repeats (CRISPR) andCRISPR-associated proteins (Cas) constitute the CRISPR-Cas system.

Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 is an endonucleasethat can be programmed by the tracrRNA/crRNA to cleave, in asite-specific manner, a DNA target sequence using two distinctendonuclease domains (HNH and RuvC/RNase H-like domains) (see U.S.Patent Application Publication No. 2014-0068797, published 6 Mar. 2014;see also Jinek, M., et al., Science, 337:816-821 (2012) and Karvelis etal. Genome Biology (2015) 16:253.

The foregoing CD163 edits, while demonstrating some indication ofefficacy against PRRSv, cannot be made as precisely and effectively asthe edits disclosed herein. There is a need to improve the health of aporcine herd by editing the CD163 gene using guides for improved editingactivity and reduced unintended edits, while conferring resistance toPRRSv.

SUMMARY

The present specification provides for and includes edited CD163 genesthat confer PRRSv resistance on pigs comprising the edited gene.

In some embodiments, the present teachings provide for and include aCD163 gene edited to confer PRRSv resistance in Sus scrofa wherein theedit excises exon 7 and the edited gene can comprise a repaired genomicsequence as set forth in any one of SEQ ID NOs: 426-458 and 520-555. Insome configurations, the edited gene can comprise a repaired genomicsequence selected from the group consisting of SEQ ID NOs: 426-458. Invarious configurations, the repaired gene sequence can comprise SEQ IDNO: 426, SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 430,SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ IDNO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439,SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO: 443, SEQ IDNO: 444, SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ ID NO: 448,SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ IDNO: 453, SEQ ID NO: 454, SEQ ID NO: 455, SEQ ID NO: 456, SEQ ID NO: 457,SEQ ID NO: 458, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ IDNO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527,SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ IDNO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536,SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ IDNO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545,SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 549, SEQ IDNO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553, SEQ ID NO: 554,or SEQ ID NO: 555. In various configurations, the edit can be createdusing a pair of gRNAs, wherein the pair of gRNAs can be SEQ ID NOs: 229and 256, SEQ ID NOs: 230 and 256, SEQ ID NOs: 231 and 256, SEQ ID NOs:237 and 256, SEQ ID NOs: 241 and 256, SEQ ID NOs: 229 and 258, SEQ IDNOs: 230 and 258, SEQ ID NOs: 231 and 258, SEQ ID NOs: 237 and 258, SEQID NOs: 241 and 258, SEQ ID NOs: 229 and 261, SEQ ID NOs: 230 and 261,SEQ ID NOs: 231 and 261, SEQ ID NOs: 237 and 261, SEQ ID NOs: 241 and261, SEQ ID NOs: 219 and 256, SEQ ID NOs: 221 and 256, SEQ ID NOs: 224and 256, SEQ ID NOs: 227 and 256, SEQ ID NOs: 219 and 258, SEQ ID NOs:221 and 258, SEQ ID NOs: 224 and 258, SEQ ID NOs: 227 and 258, SEQ IDNOs: 219 and 261, SEQ ID NOs: 221 and 261, SEQ ID NOs: 224 and 261, SEQID NOs: 227 and 261, SEQ ID NOs: 249 and 256, SEQ ID NOs: 250 and 256,SEQ ID NOs: 249 and 258, SEQ ID NOs: 250 and 258, SEQ ID NOs: 249 and261, or SEQ ID NOs: 250 and 261. In various configurations, the edit canbe created using a pair of gRNAs, wherein the pair of targeting regionscan be SEQ ID NOs: 229 and 256, SEQ ID NOs: 230 and 256, SEQ ID NOs: 231and 256, SEQ ID NOs: 241 and 256, SEQ ID NOs: 229 and 258, SEQ ID NOs:231 and 258, SEQ ID NOs: 241 and 258, SEQ ID NOs: 219 and 256, SEQ IDNOs: 221 and 256, SEQ ID NOs: 224 and 256, SEQ ID NOs: 227 and 256, SEQID NOs: 227 and 258, SEQ ID NOs: 221 and 261, SEQ ID NOs: 249 and 256,SEQ ID NOs: 250 and 256, SEQ ID NOs: 249 and 258, or SEQ ID NOs: 249 and261.

In various configurations, the edit can be created using a pair of gRNAswherein the pair of gRNAs can be SEQ ID NOs: 229 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 230 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 231 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 237 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 241 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 229 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 230 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs wherein the pair of targeting regions can be SEQ IDNOs: 231 and 258. In various configurations, the edit can be createdusing a pair of gRNAs wherein the pair of gRNAs can be SEQ ID NOs: 237and 258. In various configurations, the edit can be created using a pairof gRNAs wherein the pair of gRNAs can be SEQ ID NOs: 241 and 258. Invarious configurations, the edit can be created using a pair of gRNAswherein the pair of gRNAs can be SEQ ID NOs: 229 and 261. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 230 and 261. In variousconfigurations, the edit can be a pair of gRNAs wherein the pair ofgRNAs can be SEQ ID NOs: 231 and 261. In various configurations, theedit can be created using a pair of gRNAs wherein the pair of gRNAs canbe SEQ ID NOs: 237 and 261. In various configurations, the edit can becreated using a pair of gRNAs wherein the pair of gRNAs can be SEQ IDNOs: 241 and 261. In various configurations, the edit can be createdusing a pair of gRNAs wherein the pair of gRNAs can be SEQ ID NOs: 219and 256. In various configurations, the edit can be created using a pairof gRNAs wherein the pair of gRNAs can be SEQ ID NOs: 221 and 256. Invarious configurations, the edit can be created using a pair of gRNAswherein the pair of gRNAs can be SEQ ID NOs: 224 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 227 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 219 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 221 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 224 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 227 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 219 and 261. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 221 and 261. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 224 and 261. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 227 and 261. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 249 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 250 and 256. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 249 and 258. In variousconfigurations, the edit can be created using a pair of gRNAs whereinthe pair of gRNAs can be SEQ ID NOs: 250 and 258. In variousconfigurations, the edit can be created a pair of gRNAs wherein the pairof gRNAs can be SEQ ID NOs: 249 and 261. In various configurations, theedit can be created using a pair of gRNAs wherein the pair of gRNAs canbe SEQ ID NOs: 250 and 261. In various configurations, the repairedgenomic sequence can comprise SEQ ID NO: 453 and the edit can be createdusing sequences set forth in SEQ ID NOs: 249 and 256.

In various configurations, the instant disclosure provides for andincludes a Sus scrofa cell that can comprise the CD163 gene of thepresent teachings. In some configurations, the present teachings furtherprovide for and include a cell line that can comprise a plurality of theSus scrofa cell. In some configurations, the cell line can be afibroblast cell line. In various configurations, the cell, plurality ofcells, or cell line can be derived from PIC line 2, PIC line 3, PIC line15, PIC line 19, PIC line 27, PIC line 62, or PIC line 65. The presentteachings further provide for an embryo, piglet, or mature adult thatcan comprise a plurality of the cell.

The present disclosure also provides for a CD163 gene edited to conferPRRSv resistance in Sus scrofa wherein the edit creates a stop codonresulting in a predicted exon 7 amino acid sequence that can be selectedfrom the group consisting of 506-517. In various configurations, theamino acid sequence can be set forth in SEQ ID NO: 506, SEQ ID NO: 507,SEQ ID NO: 508, SEQ ID NO: 509, SEQ ID NO: 510, SEQ ID NO: 511, SEQ IDNO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516,or SEQ ID NO: 517. In various configurations, the predicted amino acidsequence of exon 7 can be set forth in SEQ ID NO: 513. In someconfigurations, the edit can be created using gRNAs selected from thegroup consisting of SEQ ID NOs: 351 and 365, SEQ ID NOs: 351 and 387,SEQ NOs.: 348 and 390, SEQ ID NOs: 348 and 388, SEQ ID NOs: 348 and 395,SEQ ID NOs: 352 and 365, SEQ ID NOs: 352 and 387, SEQ ID NOs: 352 and399, SEQ ID NOs: 353 and 365, SEQ ID NOs: 353 and 387, SEQ ID NOs: 353and 399, SEQ ID NOs:354 and 390, SEQ ID NOs: 354 and 388, SEQ ID NOs:354 and 395, SEQ ID NOs: 358 and 361, SEQ ID NOs: 358 and 362, SEQ IDNOs: 358 and 368, SEQ ID NOs: 358 and 384, SEQ ID NOs: 358 and 394, SEQID NOs: 358 and 399, SEQ ID NOs: 359 and 390, SEQ ID NOs: 359 and 388,SEQ ID NOs: 359 and 395, SEQ ID NOs: 360 and 368, SEQ ID NOs: 360 and384, SEQ ID NOs: 360 and 389, SEQ ID NOs: 360 and 394, SEQ ID NOs: 360and 397, SEQ ID NOs: 361 and 365, SEQ ID NOs: 361 and 387, SEQ ID NOs:362 and 390, SEQ ID NOs: 362 and 388, SEQ ID NOs: 362 and 395, SEQ IDNOs. 364 and 365, SEQ ID NOs: 364 and 387, SEQ ID NOs: 364 and 399, SEQID NOs: 365 and 368, SEQ ID NOs: 365 and 384, SEQ ID NOs: 365 and 389,SEQ ID NOs: 365 and 394, SEQ ID NOs: 365 and 397, SEQ ID NOs: 366 and368, SEQ ID NOs: 366 and 384, SEQ ID NOs: 366 and 389, SEQ ID NOs: 366and 394, and SEQ ID NOs: 366 and 397. In various configurations, theedit can be created using gRNAs selected from the group consisting ofSEQ ID NOs: 351 and 365, SEQ ID NOs: 348 and 390, SEQ ID NOs: 348 and388, SEQ ID NOs: 354 and 390, SEQ ID NOs: 358 and 394, SEQ ID NOs: 362and 390, and SEQ ID NOs: 366 and 394. In various configurations, thegRNAs used to create the edit can be SEQ ID NOs: 351 and 365. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 351and 387. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 348 and 390. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 348 and 388. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 348and 395. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 352 and 365. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 352 and 387. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 352and 399. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 353 and 365. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 353 and 387. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 353and 399. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 354 and 390. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 354 and 388. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 354and 395. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 358 and 361. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 358 and 362. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 358and 368. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 358 and 384. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 358 and 394. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 358and 399. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 359 and 390. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 359 and 388. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 359and 395. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 360 and 368. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 360 and 384. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 360and 389. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 360 and 394. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 360 and 397. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 361and 365. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 361 and 387. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 362 and 390. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 362and 388. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 362 and 395. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 364 and 365. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 364and 387. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 364 and 399. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 365 and 368. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 365and 384. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 365 and 389. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 365 and 394. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 365and 397. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 366 and 368. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 366 and 384. In variousconfigurations, the gRNAs used to create the edit can be SEQ ID NOs: 366and 389. In various configurations, the gRNAs used to create the editcan be SEQ ID NOs: 366 and 394. In various configurations, the gRNAsused to create the edit can be SEQ ID NOs: 366 and 397.

In various configurations, the edited CD163 gene can have a nucleic acidsequence selected from the group consisting of SEQ ID NO: 459-504. Invarious configurations, the repaired gene can have a nucleic acidsequence set forth in SEQ ID NO: 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,495, 496, 497, 498, 499, 500, 501, 502, 503, or 504. In variousconfigurations, the repaired gene can have a nucleic acid sequence asset forth in SEQ ID NO: 489. In various configurations the predictedamino acid sequence of exon 7 can be as set forth in SEQ ID NO: 513. Invarious configurations, the predicted amino acid sequence of the genecan be SEQ ID NO: 513, the repaired gene can have a nucleic acidsequence set forth in SEQ ID NO: 489, and the edit can be created usingsequences set forth in SEQ ID NOs: 362 and 390.

The present disclosure provides for and includes a Sus scrofa cell thatcan comprise the CD163 gene edited to comprise an exogenous stop codonas described supra. The present disclosure also provides for a cell linethat can comprise a plurality of the cell comprising the CD163 geneedited to comprise an exogenous stop codon. In some configurations, thecell line can be a fibroblast cell line. In various configurations, thecell can be derived from PIC line 2, PIC line 3, PIC line 15, PIC line19, PIC line 27, PIC line 62, or PIC line 65. The present disclosurealso provides for and includes an embryo, piglet, or mature adultcomprising a plurality of the cell that can comprise the CD163 geneedited to comprise an exogenous stop codon.

In various embodiments, the present teachings provide for and include apair of gRNAs for editing a Sus scrofa CD163 gene having a sequence asset forth in SEQ ID NOs: 229 and 256, SEQ ID NOs: 230 and 256, SEQ IDNOs: 231 and 256, SEQ ID NOs: 237 and 256, SEQ ID NOs: 241 and 256, SEQID NOs: 229 and 258, SEQ ID NOs: 230 and 258, SEQ ID NOs: 231 and 258,SEQ ID NOs: 237 and 258, SEQ ID NOs: 241 and 258, SEQ ID NOs: 229 and261, SEQ ID NOs: 230 and 261, SEQ ID NOs: 231 and 261, SEQ ID NOs: 237and 261, SEQ ID NOs: 241 and 261, SEQ ID NOs: 219 and 256, SEQ ID NOs:221 and 256, SEQ ID NOs: 224 and 256, SEQ ID NOs: 227 and 256, SEQ IDNOs: 219 and 258, SEQ ID NOs: 221 and 258, SEQ ID NOs: 224 and 258, SEQID NOs: 227 and 258, SEQ ID NOs: 219 and 261, SEQ ID NOs: 221 and 261,SEQ ID NOs: 224 and 261, SEQ ID NOs: 227 and 261, SEQ ID NOs: 249 and256, SEQ ID NOs: 250 and 256, SEQ ID NOs: 249 and 258, SEQ ID NOs: 250and 258, SEQ ID NOs: 249 and 261, SEQ ID NOs: 250 and 261, SEQ ID NOs:351 and 365, SEQ ID NOs: 351 and 387, SEQ NOs: 348 and 390, SEQ ID NOs:348 and 388, SEQ ID NOs: 348 and 395, SEQ ID NOs: 352 and 365, SEQ IDNOs: 352 and 387, SEQ ID NOs: 352 and 399, SEQ ID NOs: 353 and 365, SEQID NOs: 353 and 387, SEQ ID NOs: 353 and 399, SEQ ID NOs: 354 and 390,SEQ ID NOs: 354 and 388, SEQ ID NOs: 354 and 395, SEQ ID NOs: 358 and361, SEQ ID NOs: 358 and 362, SEQ ID NOs: 358 and 368, SEQ ID NOs: 358and 384, SEQ ID NOs: 358 and 394, SEQ ID NOs: 358 and 399, SEQ ID NOs:359 and 390, SEQ ID NOs: 359 and 388, SEQ ID NOs: 359 and 395, SEQ IDNOs: 360 and 368, SEQ ID NOs: 360 and 384, SEQ ID NOs: 360 and 389, SEQID NOs: 360 and 394, SEQ ID NOs: 360 and 397, SEQ ID NOs: 361 and 365,SEQ ID NOs: 361 and 387, SEQ ID NOs: 362 and 390, SEQ ID NOs: 362 and388, SEQ ID NOs: 362 and 395, SEQ ID NOs: 364 and 365, SEQ ID NOs: 364and 387, SEQ ID NOs: 364 and 399, SEQ ID NOs: 365 and 368, SEQ ID NOs:365 and 384, SEQ ID NOs: 365 and 389, SEQ ID NOs: 365 and 394, SEQ IDNOs: 365 and 397, SEQ ID NOs: 366 and 368, SEQ ID NOs: 366 and 384, SEQID NOs: 366 and 389, SEQ ID NOs: 366 and 394, or SEQ ID NOs: 366 and397. In some configurations, the pair of gRNAs can have a sequence asset forth in SEQ ID NOs: 229 and 256, SEQ ID NOs: 230 and 256, SEQ IDNOs: 231 and 256, SEQ ID NOs: 241 and 256, SEQ ID NOs: 229 and 258, SEQID NOs: 231 and 258, SEQ ID NOs: 241 and 258, SEQ ID NOs: 219 and 256,SEQ ID NOs: 221 and 256, SEQ ID NOs: 224 and 256, SEQ ID NOs: 227 and256, SEQ ID NOs: 227 and 258, SEQ ID NOs: 221 and 261, SEQ ID NOs: 249and 256, SEQ ID NOs: 250 and 256, SEQ ID NOs: 249 and 258, SEQ ID NOs:249 and 261, SEQ ID NOs: 351 and 365, SEQ ID NOs: 348 and 390, SEQ IDNOs: 348 and 388, SEQ ID NOs: 354 and 390, SEQ ID NOs: 358 and 394, SEQID NOs: 362 and 390, or SEQ ID NOs: 366 and 394. In variousconfigurations, the pair of gRNAs can have a sequence as set forth inSEQ ID NOs: 249 and 256. In various configurations, the pair of gRNAscan have a sequence as set forth in SEQ ID NOs: 362 and 390.

The present disclosure provides for and includes a CRISPR complex forediting a CD163 gene of Sus scrofa comprising a pair of gRNAs of thepresent teachings.

The present disclosure provides for and includes a method for editing aCD163 gene of Sus scrofa comprising using a CRISPR-CAS complexcomprising a pair of gRNAs of the present teachings.

The present disclosure provides for and includes a method for preparinga PRSSV resistant Sus scrofa cell by using a CRISPR-CAS complexcomprising the pair of gRNAs of the present teachings.

The present disclosure provides for and includes a method of producingPRRSv resistant Sus scrofa animals, comprising: a) editing a CD163 geneof a Sus scrofa cell or a plurality of Sus scrofa cells using a CRISPRcomplex comprising a pair of gRNAs of the present teachings; and b)producing an animal from the cell or plurality of cells.

The present disclosure provides for and includes the use of a cell lineaccording to the present teachings in producing PRRSv resistant animals.

The present disclosure also provides for and includes an embryo, piglet,or mature adult comprising a plurality of the cell according to thepresent teachings.

In various embodiments the present disclosure provides for and includesa method of determining the presence or absence of an edited sequencehaving 90% or 95% identity with a sequence set forth in SEQ ID NO: 453comprising performing real time PCR with a) differentially labeledprobes of sequences set forth in SEQ ID NO: 564 and SEQ ID NO: 558 or561; b) a primer pair set forth in SEQ ID NOs: 562 and 563; and c) aprimer pair set forth in SEQ ID NOs: 556 and 557 or SEQ ID NOs: 559 and560. In some configurations, the edited sequence can have 100% identitywith the sequence set forth in SEQ ID NO: 453. In some configurations,the edited sequence can have 90% identity with the sequence set forth inSEQ ID NO: 453. In some configurations, the edited sequence can have 95%identity with the sequence set forth in SEQ ID NO: 453.

In various embodiments, the present teachings provide for and include aPCR primer selected from the group consisting of SEQ ID NO: 556, SEQ IDNO: 557, SEQ ID NO: 559, SEQ ID NO: 560, SEQ ID NO: 562, and SEQ ID NO:563. In some configurations, the PCR primer can have a sequence as setforth in SEQ ID NO: 556. In various configurations, the PCR primer canhave a sequence as set forth in SEQ ID NO: 557. In variousconfigurations, the PCR primer can have a sequence as set forth in SEQID NO: 559. In various configurations, the PCR primer can have asequence as set forth in SEQ ID NO: 560. In various configurations, thePCR primer can have a sequence as set forth in SEQ ID NO: 562. Invarious configurations, the PCR primer can have a sequence as set forthin SEQ ID NO: 563.

In some embodiments, the present disclosure provides for and includes areal time PCR probe selected from the group consisting of SEQ ID NO:558, SEQ ID NO: 561, and SEQ ID NO: 564. In some configurations, theprobe has a sequence as set forth in SEQ ID NO: 558. In variousconfigurations, the probe has a sequence as set forth in SEQ ID NO: 561.In various configurations, the probe has a sequence as set forth in SEQID NO: 564.

In various embodiments, the present teachings provide for and includethe use of PCR primers set forth in a) SEQ ID NO: 556 and 557 and SEQ IDNO: 562 and 563 or b) SEQ ID NO: 559 and 560 and SEQ ID NO: 562 and 563to determine the presence or absence of an edited genome sequence setforth in SEQ ID NO: 453.

In various embodiments, the present teachings provide for and includethe use of PCR probes set forth in a) SEQ ID NOs: 558 and 564 or b) SEQID NOs: 561 and 564 to determine the presence or absence of an editedgenome sequence set forth in SEQ ID NO: 453.

In various embodiments, the present teachings provide for and include amethod of creating a PRRSv resistant pig comprising editing the pig'sgenome to comprise a genomic sequence as set forth in SEQ ID NO: 453. Insome configurations, the editing the pig's genome can compriseadministering gRNAs having sequences set forth in SEQ ID NOs: 249 and256. In various configurations, the administering comprises injectingpre-formed RNP complexes comprising the gRNAs and a CAS protein into azygote, embryo, or MII oocyte. In various configurations, the pig is aPIC™ line 2 (Pig Improvement Company, Ltd, Basingstoke, UK), PIC™ line3, PIC™ line 15, PIC™ line 19, PIC™ line 27, PIC™ line 62, or PIC™ line65 pig.

In various embodiments, the present disclosure provides for andincludes, a Sus scrofa animal comprising an edited gene that confersPRRSv resistance in Sus scrofa wherein the edit excises exon 7 and theedited gene comprises a repaired genomic sequence set forth in SEQ IDNO: 453. In some configurations, the edit can be made with guide RNAs(gRNAs) having sequences set forth in SEQ ID NOs: 249 and 256. Invarious configurations, the animal can be an edited animal of PIC™ line2, PIC™ line 3, PIC™ line 15, PIC™ line 19, PIC™ line 27, PIC™ line 62,or PIC™ line 65. In various configurations, the present disclosureprovides for and includes a cell prepared from the animal of the presentteachings. In various configurations, the present disclosure providesfor and includes a cell line prepared from the cell according to thepresent teachings. In some configurations, the cell line can be afibroblast cell line.

In some embodiments, the present teachings provide for and include aCD163 gene edited to confer PRRSv resistance in Sus scrofa wherein theedit excises exon 7 and the edited gene comprises a repaired genomicsequence set forth in SEQ ID NO: 453. In some configurations, the editis created using sequences set forth in SEQ ID NOs: 249 and 256. Invarious configurations, the present disclosure provides for a Sus scrofacell comprising the CD163 gene according to the present teachings. Insome configurations, the present disclosure provides for a cell linecomprising a plurality of the cell according to the present teachings.In some configurations, the cell line can be a fibroblast cell line. Invarious configurations, the cell can be isolated from PIC line 2, PICline 3, PIC line 15, PIC line 19, PIC line 27, PIC line 62, or PIC line65.

In various embodiments, the present disclosure provides for and includesa pair of gRNAs for editing a Sus scrofa CD163 gene comprising the guidesequences set forth in SEQ ID NOs: 249 and 256.

In various embodiments, the present teachings provide for and include amethod of creating a PRRSv resistant pig comprising editing the pig'sgenome to comprise a genomic sequence as set forth in SEQ ID NO: 453. Insome configurations, editing the pig's genome can comprise administeringgRNAs having sequences set forth in SEQ ID NOs: 249 and 256. In someconfigurations, the administering can comprise injecting pre-formed RNPcomplexes comprising the gRNAs and a CAS protein into a zygote, embryo,or MII oocyte. In various configurations, the pig can be a PIC™ line 2,PIC™ line 3, PIC™ line 15, PIC™ line 19, PIC™ line 27, PIC™ line 62, orPIC™ line 65 pig.

DETAILED DESCRIPTION

The aspects of the present teachings include, but are not limited to,particular methods of improving the health of a porcine species bytargeted inactivation of CD163, which can vary and are understood byskilled artisans. It is further to be understood that all terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting in any manner or scope. For example,as used in this specification and the appended claims, the singularforms “a,” “an,” and “the” can include plural referents unless thecontent clearly indicates otherwise. Further, all units, prefixes, andsymbols may be denoted in their SI accepted forms. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range.

So that the present application may be more readily understood, certainterms are first defined. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which aspects of thepresent application pertain. Many methods and materials similar,modified, or equivalent to those described herein can be used in thepractice of the aspects of the present application without undueexperimentation, the preferred materials and methods are describedherein. In describing and claiming the aspects of the presentapplication, the following terminology will be used in accordance withthe definitions set out below.

As used herein, “animal cell” means a cell, including, but not limitedto, a somatic cell, culture cell, gamete cell, blood cell, zygote, andembryonic cell. These animal cells can be reproductive ornon-reproductive cells. As used herein, cells may be isolated from ananimal or embryo and maintained in tissue culture. Also included aremixed cultures that can comprise gene edited cells of the presentspecification and a non-gene edited support cell or feeder cell.

As used herein, the terms “gene edited,” “genetically edited,” and“genome-edited,” refer to the use of homing technology with naturallyoccurring or artificially engineered endonucleases, often referred to as“homing endonucleases,” or “targeting endonucleases.” “Genome-editing”and “gene editing,” refer to altering the genome by deleting, inserting,or substituting specific nucleic acid sequences. The altering can begene or location specific, but need not be altering the sequence of agene per se. Genome editing can use endonucleases such as the CRISPRsystem to cut a nucleic acid, thereby generating a site for thealteration. Other endonucleases are available and are suitable for use;however, off-site cutting and specificity can be significant problems.In systems like CRISPR and others, the nuclease can be directed to thetarget site by complexing with a polynucleotide, herein called a “targetsequence,” to introduce a site specific DSB. Not to be limited bytheory, the DSB can then be repaired by endogenous non-homologous endjoining (NHEJ) machinery.

A number of endonucleases are known that are suitable for, and have beenadapted to, gene editing. Gene editing methods are known in the art,including Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) systems (e.g., the CRISPR/Cas9 system), TranscriptionActivator-Like Effector Nucleases (TALENs), and Gene editing nucleasesincluding Zinc Finger Nucleases (ZFNs). Gene edited animals can bedistinguished from transgenic animals by the incorporation of exogenousDNA sequences, particularly sequences having identity with sequencesfrom foreign species in the latter. Here, the terms include, and providefor, small deletions and insertions that do not introduce more than 10nucleotides. The terms also encompass progeny animals such as thosecreated by sexual crosses or asexual propagation from the initial geneedited animal.

As used herein, the term “repair template” or “repair sequences” refersto a polynucleotide introduced into a cell undergoing DSB repair atnuclease targeted locations in the genome to guide the repair andprovide accurate and selective editing. Repair templates can be used toselectively change or delete sequences at a locus having a DSB andgenerally comprise a 5′ genome hybridizing region and a 3′ genomehybridizing region. In an aspect, the 5′ genome hybridizing and 3′genome hybridizing regions can share at least 80% homology. In anaspect, the 5′ genome hybridizing and 3′ genome hybridizing regions canshare identity. Further descriptions of the repair templates of thepresent disclosure are provided below. For deletion templates, acontiguous region of the genome can be separated by the deletion of oneor more nucleotides. Targeted editing of individual bases can beprepared using repair templates comprising a contiguous region of agenome and having one or more base changes, including addition of a stopcodon. The percentage identity of a repair template to a contiguousregion of the genome can be between 80% and 100% using an ungappedalignment. As provided herein, a core region of identity within a repairtemplate is provided that may be flanked by a flanking homology regionsharing between 80% and 100% homology to the targeted chromosomalregion. Not to be limited by theory, the core region identity canincrease the fidelity of the repair process while extended regions ofhomology on either side increase the efficiency and allow for genotypicvariation among cells targeted for editing.

As used herein, the term “wild type” or “WT” refers to a phenotype,genotype, or gene that predominates in a natural population of pigs orline of pigs. When used to compare phenotypes and genotypes of geneedited cells and animals of specific lines, the term “wild type” refersto non-gene edited cells and animals of the same line. In an aspect, thepresent disclosure provides for comparison of edited pigs to non-editedpigs of a similar breed having a similar genetic background. In anaspect, the present disclosure provides for comparison of edited pigs tonon-edited pigs of the same breeding line.

As used herein, the term “knock-out” refers to the disruption of genefunction by reduction or elimination of its expression. Knock-outs maybe generated through the creation of double-strand breaks (DSBs) in thechromosome which can then be repaired using either non-homologous endjoining (NHEJ) or homologous recombination of DNA repair templates ortargeting vectors by homology-directed repair (HDR). Not to be limitedby theory, HDR knock-outs may also be prepared by microhomology-mediatedend joining providing a repair template to insert, delete, or editgenomic sequences. Knock-outs may also be generated through replacementvectors, or hit-and-run vectors, or random insertion of a gene trapvector resulting in complete, partial, or conditional loss of genefunction.

References herein to a deletion in a nucleotide sequence spanning arange are inclusive of all nucleotides in the listed range. For example,a 5 base pair deletion from nucleotide “a” to nucleotide “e” means thateach of nucleotides “a,” “b,” “c,” “d,” and “e” have been deleted (where“b,” “c,” and “d” are between “a” and “e”).

As used herein, the term “edit” includes alterations in the nucleotidesequence of a polynucleotide, such as, for example, a gene, coding DNAsequence (CDS), or non-coding DNA sequence, compared to the wild-typesequence. The term “edits” may include insertions, deletions,splice-donor site edits, point-edits, and the like.

As used herein, the term “clustered regularly interspaced shortpalindromic repeats” or “CRISPR” refers to a gene editing systemutilizing a CRISPR segment of genetic material, and the RNA segments andenzymes it produces, to identify and modify specific DNA sequences inthe genome of other organisms. CRISPR systems include Type I, Type II,and Type III CRISPR systems. As used herein, the term “CRISPR associatedprotein” or “Cas” refers to a protein family that can be strictlyassociated with CRISPR elements and always occurs near a repeat clusterof CRISPR segments. For example, Cas proteins can include, but are notlimited to, Cas9 family member proteins, Cas6 family member proteins(e.g., Csy4 and Cas6), and Cas5 family member proteins. Examples of Casprotein families and methods of identifying the same have been disclosedin Haft, Daniel H., et al., PLoS Comput. Bio., 2005, 1, e60.

While not being limited by any particular scientific theory, a CRISPRnuclease can form a complex with a guide RNA (gRNA), which hybridizeswith a complementary target nucleic acid molecule, thereby guiding theCRISPR nuclease to the target nucleic acid molecule. The crRNA comprisesa repeat sequence and a spacer sequence which can be complementary to aspecific protospacer sequence in an invading pathogen. It is the spacersequence that can be designed to be complementary to target sequences ina eukaryotic genome. CRISPR nucleases can associate with theirrespective crRNAs in their active forms. The present specificationprovides for, and includes a crRNA that can comprise an RNA spacercomprising the first 20 nucleotides of each of SEQ ID NOs: 22 to 271 and347 to 425. As used herein, each of SEQ ID NOs: 22 to 271 and 347 to 425provides for, and includes, the corresponding RNA sequence substitutinguridine for the thymidine and ribose for deoxyribose. Also, as usedherein, the SEQ ID NOs include the PAM (see below) which is present inthe genome sequence targeted. Skilled artisans will recognize that thegRNA targeting this sequence would not include the PAM, and thereforewould recognize that a gRNA having a sequence set forth in these SEQ IDNOs would only include the first 20 nucleotides, thus excluding the PAM.

Some CRISPR nucleases, such as CasX and Cas9, can require anothernon-coding RNA component, referred to as a trans-activating crRNA(tracrRNA), to have functional activity. A crRNA comprising a spacersequence selected from the group consisting of the first 20 nucleotidesof each of SEQ ID NOs: 22 to 271 and the first 20 nucleotides of each ofSEQ ID NOs: 347 to 425 can be covalently linked to the 5′ end of atracrRNA into one nucleic acid molecule in what is herein referred to asa “single guide RNA” (sgRNA), as described in Jinek, et al., Science,337, 2012, 816-821. As used herein, the tracrRNA is also referred to as“guide RNA backbone” or “the backbone” sequence. As used herein, gRNAincludes both a single guide RNA or separate molecules comprising thespacer sequence in the crRNA for use with a separate tracrRNA. The gRNAcan guide the active Cas complex to a target site complementary to thespacer sequence in the crRNA, where the Cas nuclease can cleave thetarget site. The crRNA can have a region of complementarity to apotential DNA target sequence and a second region that can formbase-pair hydrogen bonds with the tracrRNA to form a secondarystructure, typically to form at least one stem structure. The region ofcomplementarity to the DNA target sequence can be the spacer or spacersequence. The tracrRNA and a crRNA can interact through a number ofbase-pair hydrogen bonds to form secondary RNA structures. Complexformation between tracrRNA/crRNA and Cas9 protein can result in aconformational change of the Cas9 protein that facilitates binding toDNA, endonuclease activities of the Cas9 protein, and crRNA-guidedsite-specific DNA cleavage by the endonuclease Cas9 (Svitashev et al.,Nature Communications, 2016, 7, 113274). In practice, the 20 nucleotidesand the guide RNA backbone can be DNA (that can be expressed from apromoter to be transcribed in vivo (in cells) or in vitro (via T7polymerase) to form an RNA guide or the guide RNA backbone can bechemically synthesized dual (crRNA and trRNA) guide or single guide RNA.

For a Cas9 protein/tracrRNA/crRNA complex to cleave a double-strandedDNA target sequence, the DNA target sequence can be adjacent to acognate protospacer adjacent motif (PAM). By designing a crRNA to havean appropriate spacer sequence, the complex can be targeted to cleave ata locus of interest, e.g., a locus at which sequence modification isdesired.

A variety of Type II CRISPR-Cas system crRNA and tracrRNA sequences, andassociated predicted secondary structures are known in the art (see,e.g., Ran, F. A., et al., Nature, 2015, 520, 186-191; Fonfara et al.,Nucleic Acids Research, 2014, 42, 2577-2590). The Type II CRISPR-Cassystem of Ran et al., is provided herein in an aspect.

The spacer of Type II CRISPR-Cas systems can hybridize to a nucleic acidtarget sequence that is located 5′ or 3′ of a PAM, depending upon theCas protein to be used. A PAM can vary depending upon the Caspolypeptide to be used. For example, if Cas9 from S. pyogenes is used,the PAM can be a sequence in the nucleic acid target sequence thatcomprises the sequence 5′-NRR-3′, wherein R can be either A or G, N isany nucleotide, and N is immediately 3′ of the nucleic acid targetsequence targeted by the nucleic acid target binding sequence.Preferably, the PAM of S. pyogenes comprises 5′-NGG-3′. In anotherexample, if Cas9 from S. thermophilus CRISPR3 (Sther CR3) is used, thePAM can be a sequence in the nucleic acid target sequence that comprisesthe sequence 5′-nGGnG-3′. (See Sapranauskas et al., Nucleic AcidsResearch, 2011, 39, 9275-9282).

Other Cas proteins recognize other PAMs, and one of skill in the art candetermine the PAM for any particular Cas protein. A growing number ofCAS proteins and systems are known in the art that are suitable for usewith the targeting sequences of the present specification. See Shah etal., RNA Biol., 2013, 10, 891-899. A representative sample of CASsystems and their PAM sequences is provided in Table 1 below.

TABLE 1 Exemplary CRISPR/Cas Systems Species/Variant PAM of Cas9Sequence Streptococcus 3′ NGG Wu et al., Nat Biotechnol., 2014, 32,pyogenes (SP); 670-676 SpCas9 SpCas9 D1135E 3′ NGGNishimasu et al., Science, 2018, 361, variant (reduced 1259-1262. NAGbinding) SpCas9 VRER 3′ NGCG Nishimasu et al. (2018) variant SpCas9 EQR3′ NGAG Nishimasu et al. (2018) variant SpCas9 VQR 3′ NGAN orNishimasu et al. (2018) variant NGNG Staphylococcus 3′ NNGRRTKleinstiver et al., Nature, 2015, 523, aureus (SA); or 481-485 SaCas9NNGRR(N) Acidaminococcus 5′ TTTV Fagerlund, R., et al., 2015, Genomesp. (AsCpf1) and Biology, 16, 251 Lachnospiraceae bacterium (LbCpf1)AsCpf1 RR 5′ TYCV Nishimasu et al., Mol Cell., 2017, 67, variant 139-147LbCpf1 RR 5′ TYCV Nishimasu et al. (2017) variant AsCpf1 RVR 5′ TATVNishimasu et al. (2017) variant Neisseria 3′Hou et al., 2013, Proc. Natl. Acad. Sci. meningitidis NNNNGATUSA, 110, 15644-156449 (NM) Treponema 3′ NAAAACSun et al., Biotechnol J., 2018, 13, denticola (TD) e1700588Campylobacter NNNNRYAC Kim, et al., Nat Commun., 2017, 8, 14500.jejuni (Cj) Streptococcus NNAGAAWToth et al., 2016, Biol Direct., 11, 46 thermophilus CR1 (St)Streptococcus nGGnG Müller etal., Mol Ther., 2016, 24, 636-644thermophilus CR3

Methods that rely on any CRISPR/CAS system can have a PAM sequence ofNRR (e.g., NGG and NAA) as provided below for the S. pyogenes sequencesor can have a PAM sequence of NGGNG as provided for the S. thermophilussequences. Table 3 and the sequence listing provide the targetingsequences and chromosomal locations. It will also be appreciated bythose of skill in the art that known CAS systems having different PAMrequirements can be engineered to recognize and utilize the targetsequences disclosed herein. Examples of modifications are presented inTable 1.

The terms “CRISPR/CasN or “CRISPR/CasN system” refer to a programmablenuclease system for genetic editing that includes a CasN (e.g., Cas2,Cas5, Cas6, Cas9, etc.) protein, or derivative thereof, and one or morenon-coding RNAs that can provide the function of a CRISPR RNA (crRNA)and trans-activating RNA (tracrRNA) for the CasN. The crRNA and tracrRNAcan be used individually or can be combined to produce a “guide RNA”(gRNA). The crRNA or gRNA can provide a sequence that is complementaryto the genomic target.

The term “Cpf1” or “CAS12” refers to another programmable RNA-guidedendonuclease of a class 2 CRISPR-Cas system, described and used for geneediting purposes (Zetsche et al., Cell, 163:759-771, 2015). This systemcan use a non-specific endonuclease unit from the Cpf1 protein family,with a specificity of cleavage conferred by a single crRNA (lackingtracrRNA). Similar to Cas9, the Cpf1 coding sequence can be fused to UTRsequences described herein to improve its stability, and thus theefficiency of the resulting gene editing method.

As used herein, the terms “transcription activator-like effectornucleases” or “TALENS” refer to nucleases engineered to enable thetargeted alteration of a given DNA sequence. TALENs can comprise anon-specific DNA-cleaving nuclease fused to a TALE DNA-binding domainthat can be engineered to allow targeted gene editing. A “TALEDNA-binding domain” or “TALE” can be a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains can be involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) can be 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. A “designed”DNA binding protein can be a protein not occurring in nature whosedesign/composition results principally from rational criteria. TALENSare discussed and disclosed in Joung et al., Nat. Rev. Mol. Cell. Biol.,2013, 14, 49-55. A “selected TALE” can be a protein not found in naturewhose production results primarily from an empirical process such asphage display, interaction trap, or hybrid selection. The presentspecification provides for, and includes, the use of TALENS to the pigsdescribed herein.

“Resistance” or “disease resistance” refers to the extent to which anorganism can defend itself from and/or withstand the attack of apathogen and remain unaffected. An organism may demonstrate completeresistance, meaning it remains virtually unaffected by a pathogen.Alternatively, an organism may demonstrate partial resistance, whereinthe extent to which the pathogen affects the organisms can be less thana comparable organism with no resistance. Resistance may stem from aparticular characteristic of the organism, allowing the organism toavoid the outcome of organism-pathogen interactions. Resistance can bedemonstrated by the extent to which an organism can avoid the diseasesymptoms associated with, or reduce the incidence/severity of clinicalsigns, or reduce the clinical symptoms associated with a pathogen.

The terms “increased resistance” and “reduced susceptibility” refer to astatistically significant reduction of the incidence and/or severity ofclinical signs or clinical symptoms which are associated with infectionby a given pathogen. For example, “increased resistance” or “reducedsusceptibility” refer to a statistically significant reduction of theincidence and/or severity of clinical signs or clinical symptoms whichare associated with infection by PRRSv in an animal comprising a deletedor inactivated chromosomal sequence in a CD163 gene protein as comparedto a control animal having an unmodified chromosomal sequence. The term“statistically significant reduction of clinical symptoms” means, but isnot limited to, the frequency in the incidence of at least one clinicalsymptom in the modified group of subjects and, in some aspects, clinicalsymptoms may be statistically reduced by at least 80% lower than in thenon-modified control group after the challenge with the infectiousagent.

As used herein, the terms “reduction of the incidence and/or severity ofclinical signs” or “reduction of clinical symptoms” mean reducing thenumber of infected subjects in a group, reducing or eliminating thenumber of subjects exhibiting clinical signs of infection, or reducingthe severity of any clinical signs that are present in one or moresubjects, in comparison to wild-type infection in an otherwise similargenetic background. For example, these terms encompass any clinicalsigns of infection, lung pathology, viremia, antibody production,reduction of pathogen load, pathogen shedding, reduction in pathogentransmission, or reduction of any clinical sign symptomatic of PRRS whencompared to an otherwise similar background. Comparisons of clinicalsigns between non-edited CD163 pigs and CD163 edited pigs can beindividually or between herds. In an aspect, an individual pig does notpresent clinical signs. In an aspect, a herd of CD163 edited pigspresent reduced clinical signs. In an aspect, the size of the herd canbe at least 100 animals. In an aspect, CD163 edited pigs of the presentspecification can have reduced clinical signs of reproductive syndrome.In an aspect, a herd of CD163 edited pigs can have a reduced number ofpremature farrowings, reduced numbers of stillborn or mummified piglets,reduced numbers of PRRSv-positive piglets, or reductions in delays toreturn to service of sow. In an aspect, CD163 edited pigs of the presentspecification can have reduced clinical signs in sows and giltsincluding, but not limited to, reduced anorexia, reduced fever, reducedlethargy, reduced pneumonia, reduced agalactica, and reducedsubcutaneous and hind limb edema. In an aspect, gilts and sows that areCD163 edited pigs of the present specification can have reduced red/bluediscoloration of the ears and vulva. In an aspect, CD163 edited sows canexhibit reduced delays to return to estrus after weaning. The presentspecification provides for, and includes, reduced deaths among a herd ofCD163 edited gilts and sows. Also included and provided for by thepresent specification are reduced clinical signs in finishing pigs. Inan aspect, a herd of CD163 edited finishing pigs can have reducedrespiratory clinical signs selected from the group consisting of fever,sneezing, hyperpnoea, dyspnea, coughing, pneumonia, lethargy, periocularedema, and oculo-nasal discharge. Preferably these clinical signs can bereduced in one or more animals of the present teachings by at least 10%in comparison to subjects not having a modification in the CD163 geneand having a similar background and that become infected. In an aspect,clinical signs can be reduced in subjects of the present teachings by atleast 80%. In another aspect, clinical signs can be reduced in pigs ofthe present teachings by at least 85%. In a further aspect, clinicalsigns can be reduced in pigs of the present teachings by at least 90%.In yet another aspect, clinical signs can be reduced in pigs of thepresent teachings by at least 95%. In aspects according to the presentdisclosure, clinical signs can be reduced by 80% to 100% relative tonon-edited CD163 pigs.

The term “breeding” as used herein refers to a process comprising theselection of superior male and superior female animals use for creationof the next generation of offspring. This process further comprises theunion of male and female gametes so that fertilization occurs. Such aunion may be brought about by mating (copulation) or by in vitro or invivo artificial methods. Such artificial methods can include, but arenot limited to, artificial insemination, surgical assisted artificialinsemination, in vitro fertilization, intracytoplasmic sperm injection,zona drilling, in vitro culture of fertilized oocytes, ovary transfer,and ovary splitting. The term “breeding” as used herein also can includetransferring of a fertilized oocyte into the reproductive tract of afemale animal in order to allow for more offspring from a particularelite female.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length promoter sequence, or the complete promoter sequence. In anaspect, the reference sequence can be the Sscrofa11.1 reference genome(GenBank accession: GCA_000003025.6).

The terms “percent identity” or “percent identical” as used herein inreference to two or more nucleotide or protein sequences is calculatedby (i) comparing two optimally aligned sequences (nucleotide or protein)over a window of comparison, (ii) determining the number of positions atwhich the identical nucleic acid base (for nucleotide sequences) oramino acid residue (for proteins) occurs in both sequences to yield thenumber of matched positions, (iii) dividing the number of matchedpositions by the total number of positions in the window of comparison,and then (iv) multiplying this quotient by 100% to yield the percentidentity. If the “percent identity” is being calculated in relation to areference sequence without a particular comparison window beingspecified, then the percent identity can be determined by dividing thenumber of matched positions over the region of alignment by the totallength of the reference sequence. Accordingly, for purposes of thepresent application, when two sequences (query and subject) areoptimally aligned (with allowance for gaps in their alignment), the“percent identity” for the query sequence can be equal to the number ofidentical positions between the two sequences divided by the totalnumber of positions in the query sequence over its length (or acomparison window), which is then multiplied by 100%. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often can differ byconservative amino acid substitutions, where amino acid residues can besubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity can beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.”

For optimal alignment of sequences to calculate their percent identity,various pair-wise or multiple sequence alignment algorithms and programsare known in the art, such as ClustalW or Basic Local Alignment SearchTool (BLAST®, National Library of Medicine, Bethesda, Md.), etc., thatcan be used to compare the sequence identity or similarity between twoor more nucleotide or protein sequences. Although other alignment andcomparison methods are known in the art, the alignment and percentidentity between two sequences (including the percent identity rangesdescribed above) can be as determined by the ClustalW algorithm, see,e.g., Chenna, R., et al., Nucleic Acids Research, 2003, 31, 3497-3500;Thompson, J. D., et al., Nucleic Acids Research, 1994, 22, 4673-4680;Larkin M A et al., Bioinformatics, 2007, 23, 2947-2948; and Altschul, S.F., et al., J. Mol. Biol., 1990, 215, 403-410.

Identity to a sequence used herein can be expressed in terms of apercent identity between two sequences as determined according toalignment of the two sequences. The present specification provides forrepair template sequences that can have at least 80% identity to acontiguous region of a genome. In an aspect, the repair template canhave a 5′ region and a 3′ region sharing at least 80% identity to acontiguous region of the genome and a core region having identity to thechromosome sequences flanking the intended gene edit site.

The present specification provides for, and includes, but is not limitedto, target sequences that can be 100% identical to the target sequencesselected from the group consisting of SEQ ID NOs: 22 to 271 and 347 to425. These target sequences can comprise a PAM sequence such as thoselisted on Table 1. Accordingly, when incorporated into a guide RNA, inan aspect the spacer region can share 100% identity to a sequenceselected from the group of the first 20 nucleotides of each of SEQ IDNOs: 22 to 271 and the first 20 nucleotides of each of SEQ ID NOs: 347to 425. Also included are spacer sequences that can have from 15 to 20nucleotides of each of SEQ ID NOs: 22 to 271 or from 15 to 20nucleotides of each of SEQ ID NOs: 347 to 425. (The remainder of thesequence can comprise the PAM sequence which can be present in thegenome, but not the gRNA molecules.) Also included and provided for areguide RNA spacer sequences that can have one or more mismatches to thetarget sequence. In an aspect, the mismatch can be at the distal end ofthe sequence target sequence to ensure that identity at the endonucleasecleavage site is maintained. Typically, the mismatches can occur at the5′ end of the target sequence relative to the nuclease site at the 3′end. In an aspect, a target sequence, or the spacer sequences of a guideRNA prepared therefrom, may have a single mismatch. In another aspect, atarget sequence, or the spacer sequences of a guide RNA preparedtherefrom, may have two (2) mismatches. In another aspect, a sequence,or the spacer sequences of a guide RNA prepared therefrom, may havethree (3) mismatches. In another aspect, a sequence, or the spacersequences of a guide RNA prepared therefrom, that may have less thanfour (4) mismatches are included. In some aspects, the mismatch regionscan be limited to terminal nucleotides distal to the PAM sequences andcleavage site.

In an aspect, the target sequences and the spacer sequence of the gRNAguide can have at least 90% identity to a sequence selected from thegroup consisting of the first 20 nucleotides of each of SEQ ID NOs: 22to 271 and the first 20 nucleotides of each of SEQ ID NOs: 347 to 425.In a further aspect, the RNA guide can have at least 90% identity and100% identity at 15 nucleotides at the 3′ end of the sequence. In anaspect, the target sequences and the RNA guide can have at least 95%identity. In a further aspect, the RNA guide can have at least 95%identity or 100% identity at 15 nucleotides at the 3′ end of thesequence. In an aspect, the target sequences and the RNA guide can haveat least 96% identity. In a further aspect, the RNA guide can have atleast 96% identity or 100% identity at 15 nucleotides at the 3′ end ofthe sequence. In an aspect, the target sequences and the RNA guide canhave at least 97% identity. In a further aspect, the RNA guide can haveat least 97% identity or 100% identity at 15 nucleotides at the 3′ endof the sequence. In an aspect, the target sequences and the RNA guidecan have at least 98% identity. In a further aspect, the RNA guide canhave at least 98% identity or 100% identity at 15 nucleotides at the 3′end of the sequence. In an aspect, the target sequences and the RNAguide can have at least 99% identity. In a further aspect, the RNA guidecan have at least 99% identity or 100% identity at 15 nucleotides at the3′ end of the sequence. In an aspect, the target sequences and the RNAguide can have at least 100% identity. Also included and provided for bythe present specification are spacer sequences that can comprise thefirst 15 nucleotides each of SEQ ID NOs: 22 to 271 or the first 15nucleotides each of SEQ ID NOs: 347 to 425. In an aspect, the spacersequences can comprise the first 16 nucleotides in each of SEQ ID NOs:22 to 271 or the first 16 nucleotides in each of SEQ ID NOs: 347 to 425.In another aspect, the spacer sequences can comprise the first 17nucleotides in each of SEQ ID NOs: 22 to 271 or the first 17 nucleotidesin each of SEQ ID NOs: 347 to 425. In an aspect, the spacer sequencescan comprise the first 18 nucleotides in each of SEQ ID NOs: 22 to 271or the first 18 nucleotides in each of SEQ ID NOs: 347 to 425. In yetanother aspect, the spacer sequences can comprise the first 19nucleotides in each of SEQ ID NOs: 22 to 271 or the first 19 nucleotidesin each of SEQ ID NOs: 347 to 425.

The preferred meaning of “fertilization” as used herein to refer to pigbreeding encompasses any technique that produces a viable embryo.Fertilization can include both insemination of female pigs, and in vitroor ex vivo fertilization. There are three commonly available ways toinseminate a sow, namely traditional artificial insemination (AI),intrauterine insemination (IUI), and deep intrauterine insemination(DIUI). These techniques all rely on providing a dose of semen,typically fresh and unfrozen, for insemination. In vitro fertilization(IVF) can be the harvesting of unfertilized oocytes(s) and thesubsequent fertilization of those oocytes with semen in vitro (i.e., inthe laboratory) instead of in vivo (i.e., in the live animal;insemination discussed above) as in standard ET. The fertilizedoocytes(s) or embryo(s) from the oocyte donor can then be transferredinto another female (embryo recipient). Embryo transfer (ET) can be theharvesting of fertilized oocytes(s) or embryo(s) from one female (embryodonor) and transfer of those embryo(s) into another female (embryorecipient) whose reproductive status can be synchronized with that ofthe donor.

As described in Cameron et. al. (Nat Methods, 2017, 14, 600-606), theCRISPR-Cas9 system can be used for genome editing in both basic researchand biotechnology. The application of CRISPR and related technologies togene or genome editing can be accompanied by unwanted off-targetcleavage activity and possible pathogenic and other negative phenotypicconsequences. To minimize off-target cleavage, a variety of genome-wideexperimental methods have been recently developed but some methods arepotentially biased due to cellular context or inefficiencies inrecovering relevant cleavage sites. On approach, the SITE-SEQ® assay(Caribou Biosciences, Inc., Berkeley Calif.) disclosed in Cameron et.al. enables one to comprehensively list Cas9 cleavage sites in a samplegenome, then probe those sites for cellular off-target editing infollow-up experiments through (1) extraction and purification ofhigh-molecular weight genomic DNA, (2) execution of Cas9Ribonucleoprotein (RNP) cleavage in vitro, (3) fragmentation, adapterligation, and affinity purification to enrich for Cas9 cleavedfragments, and (4) amplification and indexing of SITE-SEQ® libraries forILLUMINA® (ILLUMINA®, San Diego, Calif.) sequencing. (See Cameron etal., Nat Methods, 2017, 14, 600-606.) Various other methods ofdiscovering and reducing the number of potential off-target edits areknown in the genome editing arts. While such bioinformatics tools can beinvaluable for identifying suitable target sequences, the error rate isgenerally higher than desired and the approach can be limited when thesystem lacks extensive sequence data. Further, the fidelity of DSB breakrepair can vary, thus confounding the ability to predict and preventunwanted off target effects. Further testing of in silico selectedtarget sequences can identify select sequences that have higherefficiencies and fewer off target cuts. Testing in cell culture systemscombined with high-throughput sequencing methods can be used to identifypoor performing target constructs.

For example, the type II CRISPR system, which is derived fromStreptococcus pyogenes (S. pyogenes), can be reconstituted in mammaliancells using Cas9, a specificity-determining CRISPR RNA (crRNA)comprising a backbone sequence and a sequence selected from the groupconsisting of the first twenty nucleotides of each of SEQ ID NOs: 22 to271 and the first twenty nucleotides of each of SEQ ID NOs: 347 to 425,and an auxiliary trans-activating RNA (tracrRNA). The term “off targeteffect” broadly refers to any impact distinct from and not intended as aresult of the on-target treatment or procedure. Examples of off-targetedits can include double strand breaks at unintended locations that leadto DNA insertions of unintended nucleotides or repair templates,deletions, or rearrangements. For some systems, for example the S.pyogenes and S. thermophilus CRISPR system, the crRNA and tracrRNAduplexes can be fused to generate a single-guide RNA (sgRNA). The first20 nucleotides of the sgRNA can be complementary to the target DNAsequence and can be the spacer region, and those 20 nucleotides can befollowed at the 3′ end by a protospacer adjacent motif (PAM) in thegenome (but not the guide). In an aspect, the first 20 nucleotides ofthe sgRNA and PAM sequence can be a sequence selected from the groupconsisting of SEQ ID NOs: 22 to 271 and 347 to 425. Accordingly, asprovided below at Table 2 and SEQ ID NOs: 22 to 271 and 347 to 425,specific targeting of the Cas nuclease from either S. pyogenes or S.thermophilus can be accomplished combining the crRNAs with a backbonesequence. Although the 20-nucleotide guide sequence plus PAM sequence(e.g., 23 to 25 nucleotides) of the sgRNA can provide tightly controlledtargeting and cleavage, it has been discovered that off-target cleavageactivity can still occur on DNA sequences with between 3-5 base pairmismatches in the PAM-distal part of the sgRNA-guiding sequence.Further, different types of guide RNA structures actually affect thecleavage precision, increasing or decreasing cleave on off-target sites.Various techniques, as well as a further description of off-targetcleavage, are reviewed in Zhang et al., Mol Ther Nucleic Acids, 2015, 4,e264. The mechanisms and effects of off-target cleavage are still poorlyunderstood, meaning it can be difficult to predict and to compensate forits effects. However, the consequences can be severe; off-targetcleavage can ultimately lead to genomic instability and disrupt thefunctionality of otherwise normal genes.

The present specification provides for, and includes, pigs that can haveinactivating edits in both alleles of the CD163 gene and that can beresistant to infection with PRRSv. The CD163 pigs disclosed hereinfurther do not comprise new sequences or polypeptides, nor do theycomprise non-native amino acids resulting from frameshifts or missensemutations.

The gene edits disclosed herein allow for the protection ofCD163-positive fetuses (e.g., fetuses that have one or two wild-typeCD163 alleles). CD163-positive fetuses can be protected from PRRSvinfection while in utero so long as the dam possesses inactivating editsin both alleles of her CD163 genes (PCT/US2018/027944). For example,dams having inactivating edits in both alleles of the CD163 gene can bemated with males having two wild-type CD163 alleles, and the resultingheterozygous fetuses will be protected from PRRSv infection in utero.

In an aspect, pigs having inactivating edits in one allele of CD163 canbe generated using the methods of the present specification. These pigshaving heterozygous CD163 alleles (one edited, one wild-type) can bebred with other pigs also having heterozygous CD163 alleles orhomozygous edited CD163 alleles, and offspring from this breeding havinghomozygous edited CD163 alleles can be selected for resistance toinfection with PRRSv. The present disclosure also provides for, andincludes, porcine animals and populations, and methods for creating orimproving porcine animals and populations, in which the animals can behomozygous for one or more particular genetic markers or alleles. Invarious aspects, the present disclosure provides for, and includes,animals, and methods for creating or improving crossing porcine animalsand populations, by generating animals that can be heterozygous for oneor more particular genetic markers or alleles, and crossing said animalswith other animals heterozygous for one or more of the particulargenetic markers or alleles to produce animals that can be homozygous forthe one or more particular genetic markers or alleles. In some aspects,multiple rounds of crossing and/or back-crossing may be required toobtain homozygosity for each of the particular genetic markers andalleles. In other aspects, a single cross may be sufficient to obtainhomozygosity.

Various techniques known in the art can be used to inactivate genes tomake knock-out animals and/or to introduce edited genes into animals toproduce founder animals and to make animal lines, in which the knockoutor nucleic acid construct can be integrated into the genome. Suchtechniques can include, without limitation, pronuclear microinjection(U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germlines (Van der Putten et al., Proc. Natl. Acad. Sci. USA, 1985, 82,6148-1652), gene targeting into embryonic stem cells (Thompson et al.,Cell, 1989, 56, 313-321), electroporation of embryos (Lo, Mol. Cell.Biol., 1983, 3, 1803-1814), sperm-mediated gene transfer (Lavitrano etal., Proc. Natl. Acad. Sci. USA, 2002, 99, 14230-14235; Lavitrano etal., Reprod. Fert. Develop., 2006, 18, 19-23), and in vitrotransformation of somatic cells, such as cumulus or mammary cells, oradult, fetal, or embryonic stem cells, followed by nucleartransplantation (Wilmut et al., Nature, 1997, 385, 810-813 and Wakayamaet al., Nature, 1998, 394, 369-374). Pronuclear microinjection, spermmediated gene transfer, and somatic cell nuclear transfer can be otheruseful techniques. An animal can be genomically edited in that all ofits cells have the edit, including its germ line cells. When methods areused that produce an animal that is mosaic in its modification, theanimals may be mosaic and can be further inbred and progeny that aregenomically edited can be produced and selected using standard methods.Cloning, for instance, may be used to make a mosaic animal if its cellsare edited at the blastocyst stage, or genomic modification can takeplace when a single-cell is edited. In an aspect, an inactivatedknock-out edit can be homozygous.

In an embryo/zygote microinjection aspect, a nucleic acid construct,mRNA, protein, polynucleotides, or combinations thereof, can beintroduced into a fertilized egg. In an aspect, one or two cellfertilized eggs can be used. One and two cell fertilized eggs canprovide a visible nuclear structure containing the genetic material fromthe sperm head and the egg within the protoplasm. In an aspect,pronuclear staged fertilized eggs can be obtained in vitro or in vivo(i.e., surgically recovered from the oviduct of donor animals). Inanother aspect, in vitro fertilized eggs can be produced, for example,from collected swine ovaries by follicles aspirated using methods knownin the art. See, e.g., Agung et al., J Reprod Dev., 2013, 59, 103-106and Appeltant et al., J Reprod Dev., 2016, 62, 439-449. In an aspect,mature oocytes can be provided for use in the in vitro fertilizationmethods. In another aspect, mature oocytes can be injected with theCRISPR/Cas gene editing system of the present specification.

The present specification further provides for in vitro fertilization ofmature oocytes. In an aspect, the oocytes can be matured in vitro asprovided above. In another aspect, mature oocytes can be collected fromgilts. In vitro fertilization is performed according to establishedmethods. See Appeltant et al., J Reprod Dev., 2011, 57, 9-16, and Fowleret al., Reprod Biol., 2018, 18, 203-211.

Zygotes or embryos can be obtained for germline editing by artificialinsemination and flushing. The collected zygotes or embryos can then beedited using the methods provided herein. In an aspect, linearizednucleic acid constructs, mRNA, proteins, polynucleotides or combinationsthereof can be injected or otherwise introduced, for example, byelectroporation, into one of the pronuclei or into the cytoplasm of azygote or embryo post-fertilization, or into a gamete cellpre-fertilization. In an aspect, a pre-formed RNP complex comprising theCas nuclease protein and a guide RNA comprising a backbone of SEQ ID NO:19 and a targeting sequence selected from any one of SEQ ID NOs: 22 to271 and 347 to 425 can be prepared and injected into the embryo, zygote,or oocyte. In another aspect, a pre-formed RNP complex comprising theCas nuclease and a guide RNA comprising a backbone of SEQ ID NO: 19 anda spacer sequence selected from an RNA sequence of a sequence selectedfrom the group consisting of the first 20 nucleotides of each of SEQ IDNOs: 22 to 271 can be prepared and injected into the embryo, zygote, oroocyte, together with a repair template comprising SEQ ID NOs: 1 to 13listed in Table 6. Also included, and provided for, by the presentspecification are the combinations of spacer sequences and repairtemplates that can have the sequences of SEQ ID NOs:1 to 13 as recitedin Table 6. In an aspect, the injected zygotes or embryos can betransferred to a recipient female (e.g., into the oviducts of arecipient female) and allowed to develop in the recipient female toproduce the transgenic or gene edited animals. In an aspect, the methodsfurther provide for in vitro or in vivo fertilized zygotes or embryosthat can be centrifuged at 15,000×g for 5 minutes to sediment lipidsallowing visualization of the pronucleus. The zygotes or embryos can beinjected with using an EPPENDORF® FEMTOJET® (EPPENDORF® AG, Germany)injector and can be cultured until blastocyst formation. Rates of embryocleavage and blastocyst formation and quality can be recorded.

In an aspect, the CRISPR/Cas editing system and gRNA can be provided asa nucleic acid construct. In another aspect, the Cas nuclease may beprovided as an mRNA together with a guide RNA comprising a backbonesequence and a targeting sequence selected from the group consisting ofthe first 20 nucleotides of each of SEQ ID NOs: 22 to 271 and the first20 nucleotides of each of SEQ ID NOs: 347 to 425. In another aspect, theCas nuclease may be provided as an mRNA together with a guide RNAcomprising a backbone sequence and a targeting sequence selected fromthe group consisting of the first 20 nucleotides of each of SEQ ID NOs:22 to 271 together with a repair template listed in Table 6 and selectedfrom the group consisting of SEQ ID NOs: 1 to 13. In a further aspect,the gene targeting complex can be provided by microinjection of atranscribable DNA encoding a Cas nuclease and a guide RNA comprising abackbone sequence and a targeting sequence selected from the groupconsisting of the first 20 nucleotides of each of SEQ ID NOs: 22 to 271and the first 20 nucleotides of each of SEQ ID NOs: 347 to 425. Inaspects according the present specification, the backbone sequence canbe a sequence of SEQ ID NO: 19. Also included and provided for by thepresent specification are the combinations of targeting sequences andrepair templates that can have one of the sequences recited in SEQ IDNOs: 1 to 13. It would be understood by persons of skill in the art thatvarious combinations of nuclease, gRNA, and optionally repair templatesequences, can be introduced into oocytes, zygotes, blastula, andembryos to achieve the goals of the present methods. It will be furtherunderstood that other backbone sequences can be used in conjunction withthe targeting sequences of SEQ ID NOs: 22 to 271 and SEQ ID NOs: 347 to425 for use with other Cas nucleases. Any Cas nuclease having a PAMsequence of NGG or NGGNG can be suitable for the preparation of abackbone sequence for combination with the targeting sequences of thepresent specification. In various aspects, the desired edit can have afinal genomic sequence of SEQ ID NOs: 426 to 505. In someconfigurations, the desired edit lacks exon 7 and can have a finalnucleotide sequence as set forth in SEQ ID NOs: 426 to 458. In variousconfigurations, the final genomic sequence can include an exogenous stopcodon and can have a final genomic sequence as set forth in SEQ ID NOs:459-504. In various configurations, these partial CD163 genes can havean exon 7 amino acid sequence as set forth in SEQ ID NOs: 506 to 517.

In an aspect, the injected zygotes or embryos can be transferred to arecipient female (e.g., into the oviducts of a recipient female) andallowed to develop in the recipient female to produce the transgenic orgene edited animals. In an aspect, the methods further provide for invitro or in vivo fertilized zygotes or embryos that can be centrifugedat 15,000×g for 5 minutes to sediment lipids allowing visualization ofthe pronucleus. The zygotes or embryos can be injected using, forexample, an EPPENDORF® FEMTOJET® injector and can be cultured untilblastocyst formation. Rates of embryo cleavage and blastocyst formationand quality can be recorded.

The present specification provides for, and includes, a recipient sowhaving one or more embryos that can be injected with a CRISPR/Cas/gRNAcombination according to the present disclosure. In an aspect, apre-formed RNP complex can be provided comprising the Cas nuclease and aguide RNA comprising a backbone of SEQ ID NO: 19 and a spacer sequenceselected from the group consisting of the first 20 nucleotides of eachof SEQ ID NOs: 22 to 271 and the first 20 nucleotides of each of SEQ IDNOs: 347 to 425. In various aspects, two pre-formed RNP complexes can beprovided comprising the Cas nuclease and a guide RNA comprising abackbone of SEQ ID NO: 19 and each comprising a different spacersequence selected from the group consisting of the first 20 nucleotidesof each of SEQ ID NOs: 22 to 271 and the first 20 nucleotides of each ofSEQ ID NOs: 347 to 425. Also provided for, and included, are recipientsows that can have one or more embryos injected with a pre-formed RNPcomplex that can comprise the Cas nuclease and a guide RNA comprising atargeting sequence selected from the group consisting of the first 20nucleotides of each of SEQ ID NOs: 22 to 271 and the first 20nucleotides of each of SEQ ID NOs: 347-425. In an aspect, the pre-formedRNP complex can be prepared and injected into the embryo, zygote, oroocyte, together with a repair template listed in Table 6 (SEQ ID NOs: 1to 13). In an aspect, a recipient sow can be a sow of a different linethan the donor embryos.

Embryos or zygotes can be surgically transferred into uteri ofsynchronous recipients. Typically, 100-200 (or 150-200) embryos orzygotes can be deposited into the ampulla-isthmus junction of theoviduct using a catheter. After surgery, real-time ultrasoundexamination of pregnancy can be performed. In an aspect, the presentspecification can include a recipient sow having 1 to 100, but moretypically 30 to 60 CRISPR/Cas/gRNA combination treated embryos whereinsaid embryos can comprise a gene edited CD163 gene comprising a sequenceof SEQ ID NOs: 1 to 18 or 426 to 505. In an aspect, the transferredembryos can comprise a mosaic of edited cells. Also included are CD163gene edited embryos that can be non-mosaic.

Methods of improving the health of a livestock animal or herd oflivestock can comprise modifying a target sequence in the genome of ananimal cell to form a truncated CD163 polypeptide. Also provided for,and included, are improved animals that can have truncated CD163polypeptides. In some aspects, the predicted truncated polypeptides donot include non-native amino acids. Without being bound by theory, thetruncated gene product can be rapidly digested in vivo by cellularproteases. In an aspect of the present specification, the truncatedCD163 can result in the complete elimination of the protein. In afurther aspect, the truncated CD163 can be non-detectable in vivo. In anaspect, the truncated CD163 can be non-detectable by immunofluorescencelabelling and FACS analysis. In yet another aspect, the truncated CD163can be non-detectable when using any of the expression analysis methodsprovided in the specification, including those provided in Example 5. Inyet another aspect, the truncated CD163 can be non-detectable when usingany of the expression analysis methods provided in the specification,including those provided in Example 8.

In another aspect, a gene edited CD163 pig can comprise a truncatedCD163 gene with no more than 25 non-native amino acids. Not to belimited by theory, truncated CD163 proteins can be destabilized andtargeted for degradation within the cell. Accordingly, CD163 proteinsequences and CD163 polypeptides comprising non-native amino acids canbe non-detectable. In another aspect, the predicted truncated CD163protein can comprise no more than 211 amino acids of the native protein.In an aspect, the truncated CD163 protein can be predicted to compriseno more than 144 amino acids of the native protein. In an aspect, thetruncated CD163 protein can be predicted to comprise no more than 133amino acids of the native protein. In yet another aspect, the truncatedCD163 protein can be predicted to comprise no more than 129 amino acidsof the native protein. In an aspect, the truncated CD163 protein can bepredicted to comprise no more than 113 amino acids of the nativeprotein. In yet another aspect, the truncated CD163 protein can bepredicted to comprise no more than 108 amino acids of the nativeprotein. In a further aspect, the truncated CD163 protein can bepredicted to comprise no more than 93 amino acids of the native protein.In yet another aspect, the truncated CD163 protein can be predicted tocomprise no more than 74 amino acids of the native protein. As providedherein, truncated CD163 proteins can comprise between 32 to 211 aminoacids of native CD163 polypeptide sequences and no more than 25non-native amino acids and can be undetectable in cells and cellextracts.

In other aspects, altered CD163 protein can have a truncation with asingle amino acid substitution. In some aspects, the altered CD163protein can have a predicted amino acid sequence of no more than 1,010amino acids.

The present specification provides for, and includes, gene edited CD163pigs that can have truncations of the CD163 protein (e.g., amino acids 1to 40 of CD163 reference sequence NP 999141). Not to be limited bytheory, it is believed that truncations within the signal peptide (alsoknown as the signal sequence) can result in a failure of the protein totranslocate to the cellular membrane and the polypeptide is subsequentlydegraded within the cell. Accordingly, CD163 proteins having signalpeptide truncations can result in CD163 null animals that are resistantto infection by PRRSv. In an aspect, the gene edited CD163 animalshaving proteins truncated in the signal sequence can comprise no morethan 25 amino acids of non-native amino acid sequences. In an aspect,the gene edited pigs can comprise no more than the first 39 amino acidsof the native CD163 protein. In an aspect, the gene edited pigs cancomprise no more than the first 39 amino acids of the native CD163protein and no more than 15 non-native amino acids. Also included, andprovided for, by the present specification are gene edited pigs that canbe predicted to comprise no more than the first 38 amino acids of thenative CD163 protein. In an aspect, the gene edited pigs can comprise nomore than the first 38 amino acids of the native CD163 protein and nomore than 15 non-native amino acids. Also included, and provided for, bythe present specification are gene edited pigs that can be predicted tocomprise no more than the first 36 amino acids of the native CD163protein. In an aspect, the gene edited pigs can comprise no more thanthe first 36 amino acids of the native CD163 protein and no more than 15non-native amino acids. Truncated proteins can be non-detectable usingmethods known to those of skill in the art.

In an aspect, the gene edited pigs can be predicted to comprise no morethan the first 34 amino acids of the native CD163 protein and no morethan 15 non-native amino acids. In an aspect, the gene edited pigs canbe predicted to comprise no more than the first 32 amino acids of thenative CD163 protein and no more than 15 non-native amino acids.Truncated proteins can be non-detectable using methods known to those ofskill in the art.

Further, the present disclosure provides for and includes truncations ofthe CD163 protein that can include deletion of exon 7 (SEQ ID NOs:426-458), sequences where an exogenous stop codon is introduced intoexon 7 (SEQ ID NO: 459-504). These truncations can include CD163proteins comprising no more than 1,010 amino acids.

Breeding techniques can be used to create animals that are homozygousfor the inactivated gene from the initial homozygous or heterozygousfounder or other heterozygous animals. Homozygous animals can begenerated. Gene edited pigs described herein can be bred with otheredited or wild-type pigs of interest to ultimately generate pigs thatare homozygous or heterozygous for the edited gene. In an aspect, thehomozygous animal can be an animal of any one of the lines generated bycrosses with PIC™ elite porcine lines 2, 3, 15, 19, 27, 62, 65, andcombinations thereof. In an aspect, the homozygous animal can be ahybrid animal prepared by a cross between animals of Line 2 and Line 3.

Gene editing has been used to address various diseases, including PRRS,in swine. While there have been several gene editing technologiesdeveloped over the past 15 years, generally these platforms can bedesigned to introduce a double-stranded break at a specific region of agenome. The introduced break can then be repaired by the cell's ownmachinery.

One repair pathway, non-homologous end-joining (NHEJ), is evolutionarilyconserved throughout all kingdoms of life and is the predominantdouble-strand break repair pathway in mammalian cells. This repairprocess can result in the random insertion or deletion of nucleotidesacross the cut site. As a practical matter, this incomplete fidelity canbe used to advantage by providing in trans a DNA repair template. In anaspect, a DNA repair template carrying an alternative allele of thetargeted site (for example, a single base polymorphism or a single ormultiple base insertion or deletion) can be co-delivered with the geneediting reagents, and through the use of the cell's homologousrecombination machinery, the DNA repair template can direct the repairto generate a new allele. In aspects according to the presentspecification, DNA repair templates can be designed and selected basedon the location of high efficiency of CRISPR/CAS cleavage (e.g., highediting frequency from Table 3) and the ability to introduce deletionsthat can result in truncated proteins that comprise only wild typesequences. Repair templates can increase the efficiency and accuracy ofgene editing. DNA repair templates suitable for the methods of thepresent application can include SEQ ID NOs: 1 to 13. In an aspect, theDNA repair templates can be paired with a guide sequence as describedbelow in Table 6. Unlike transgenesis, genome editing according to thepresent specification does not result in the introduction of foreign DNAsequences into the genome.

However, as these exogenously added DNA repair templates have thepossibility to randomly integrate in the genome, it is also advantageousto identify and use guide pairs that result in the deletion of DNAsequences such that the joined ends can result in the generation of anin-frame translational stop codon across the joined ends; when the cutsites of two guides are repaired by NHEJ in an end-to-end manner, thisnew DNA sequence, when transcribed into mRNA and translated into proteincould terminate the production of the CD163 protein. Although functionof this terminated CD163 protein can be lost, often, but not always,premature termination of protein synthesis can result in an unstablepolypeptide that can be degraded and not detectable by standard methods.Guide pairs that cut exon 7 of the CD163 gene such that the joined endsform an exogenous stop codon can include SEQ ID NOs: 351 and 365, SEQ IDNOs: 351 and 387, SEQ NOs: 348 and 390, SEQ ID NOs: 348 and 388, SEQ IDNOs: 348 and 395, SEQ ID NOs: 352 and 365, SEQ ID NOs: 352 and 387, SEQID NOs: 352 and 399, SEQ ID NOs: 353 and 365, SEQ ID NOs: 353 and 387,SEQ ID NOs: 353 and 399, SEQ ID NOs: 354 and 390, SEQ ID NOs: 354 and388, SEQ ID NOs: 354 and 395, SEQ ID NOs: 358 and 361, SEQ ID NOs: 358and 362, SEQ ID NOs: 358 and 368, SEQ ID NOs: 358 and 384, SEQ ID NOs:358 and 394, SEQ ID NOs: 358 and 399, SEQ ID NOs: 359 and 390, SEQ IDNOs: 359 and 388, SEQ ID NOs: 359 and 395, SEQ ID NOs: 360 and 368, SEQID NOs: 360 and 384, SEQ ID NOs: 360 and 389, SEQ ID NOs: 360 and 394,SEQ ID NOs: 360 and 397, SEQ ID NOs: 361 and 365, SEQ ID NOs: 361 and387, SEQ ID NOs: 362 and 390, SEQ ID NOs: 362 and 388, SEQ ID NOs: 362and 395, SEQ ID NOs: 364 and 365, SEQ ID NOs: 364 and 387, SEQ ID NOs:364 and 399, SEQ ID NOs: 365 and 368, SEQ ID NOs: 365 and 384, SEQ IDNOs: 365 and 389, SEQ ID NOs: 365 and 394, SEQ ID NOs: 365 and 397, SEQID NOs: 366 and 368, SEQ ID NOs: 366 and 384, SEQ ID NOs: 366 and 389,SEQ ID NOs: 366 and 394, and SEQ ID NOs: 366 and 397.

In one aspect, the present teachings can involve a simple, precise andreproducible single CD163 loss of function edit in elite pigs such thatthe edit occurs early in the CD163 gene. A single guide RNA-Cas proteincombination and DNA repair template can be selected for directing aspecific gene edit in elite pigs, based on several considerations andapproaches: efficient cutting near the 5′ end of the CD163 gene intissue culture, bioinformatic review identifying guides with fewmismatches in the pig genome, high specificity of on-target cutting asdetermined by biochemical prescreen, and the ability to target specificgene edits at an intended site in pig embryo-like cells. Examples ofspecific gene edits of CD163 are discussed in detail below. In anaspect, a specific gene edit according to the present specification caninclude a CD163 gene comprising a sequence selected from the groupconsisting of SEQ ID NOs: 1 to 18 and 426 to 505. In an aspect, cellscomprising an edited CD163 gene can comprise at least one allele havinga sequence selected from the group consisting of SEQ ID NOs:1 to 18 and426 to 505. In an aspect, cells comprising two gene edited alleles ofthe CD163 gene can comprise, at both alleles, a sequence selected fromthe group consisting of SEQ ID NOs: 1 to 18 and 426 to 505. In anaspect, both gene edited CD163 genes can comprise the same sequenceselected from the group consisting of SEQ ID NOs: 1 to 18 and 426-505(e.g., two alleles of SEQ ID NO: 1 or two alleles of SEQ ID NO: 2,etc.). In an aspect, the genome of a cell can comprise gene edited CD163genes comprising one each of a sequence selected from the groupconsisting of SEQ ID NOs:1 to 18 and 426-505 (e.g., an allele of SEQ IDNO: 1 with an allele of SEQ ID NO: 2, and all combinations thereof).

Also provided for, and included, are mixtures of cells that can compriseCD163 edited cells and non-gene edited cells. In an aspect, the mixturecan be an embryo. In another aspect, the mixture can be a cell culture.In a further aspect, the mixture of cells does not comprise areproductive cell. In an aspect, the present specification can providefor, and includes, a tissue culture of CD163 edited cells and methods toprepare such cultures. Cultures according to the present specificationcan include mixtures of CD163 edited cells (e.g., cells having an alleleof SEQ ID NO: 1 with cells having an allele of SEQ ID NO: 2, cellshaving an allele of SEQ ID NO: 1 with cells having an allele of SEQ IDNO: 3, and all combinations thereof). In an aspect, the tissue cultureof non-reproductive cells can include cells comprising a single CD163edit. In a further aspect, cells of a single CD163 edit in culture maycomprise one or both edited alleles.

Specific examples of endonuclease and guide RNA backbone sequences thatcan be used in the products and methods of the present teachings arelisted in Table 2.

TABLE 2 Endonuclease and Guide Backbone Sequences Sequence Identity SEQID NO: Guide RNA Backbone (DNA sequence shown) 19 Streptococcus pyogenesendonuclease protein 20 sequence Streptococcus thermophilus CR3 21endonuclease protein sequence

Elite porcine nucleus lines can be sequenced and aligned against apublic reference for the CD163 gene. These data can be used for thedevelopment of gene editing reagents for the PRRS-resistance project.These sequences can be scanned for the presence of conserved RNA-guidedCRISPR-Cas9 recognition sites which can consist of the 3 nucleotide or 5nucleotide motif, nGG (AGG, CGG, TGG, GGG), nGGnG (AGGTG, CGGTG, TGGTG,GGGTG, AGGGG, CGGGG, TGGGG, GGGGG, AGGAG, CGGAG, TGGAG, GGGAG, AGGCG,CGGCG, TGGCG, GGGCG), respectively. This motif, called the PAM sequence,can be located adjacent to, and 3′ of, a 20 nucleotide spacer sequencewhich can be used for base-pairing with the RNA. Suitable sites can beidentified, and crRNA guide or single guide sequences can be prepared,and are presented below in Table 3.

While not limited to any particular theory, when complexed, theguideRNA-Cas9 protein can recognize a DNA site for cleavage, which canthen be repaired by cellular components by either non-homologous endjoining (random repair) or by a DNA template repair pathway (homologydirected repair, HDR).

Guide RNAs (gRNA) can be generated across exons 1 to exons 7 of CD163.Edits in the virus binding domain (Domain 5) of the CD163 protein caninhibit the ability of the virus to bind to the protein, thus preventinguptake of the virus into pig lung macrophages. When the edit in CD163 ishomozygous in pigs, the animals can be resistant to PRRS infection. Thepresent specification provides for, and includes, gRNA sequencesincluding the crRNA sequences listed in Table 3 that can be selectedfrom the group consisting of the first 20 nucleotides of each of SEQ IDNOs: 22 to 271 and 347 to 425. SEQ ID NOs: 22 to 271 and 347 to 425 areshown as DNA sequences. The corresponding RNA counterpart, and thepresence of the PAM motif which is generally not included in gRNAs,would be apparent to a person of skill in the art. Also provided herein,are RNA sequences that can comprise sequences selected from the groupconsisting of the first 20 nucleotides of each of SEQ ID NOs: 22 to 271and 347 to 425. SEQ ID NOs: 22 to 271 and 347 to 425 can be combinedwith the gRNA backbone sequence and expressed as RNA for use in thepreparation of a gRNA and then mixed with the Cas nuclease to prepare anactive ribonucleotide protein complex (sgRNP). The sgRNP complex canthen be injected into a mature oocyte, a zygote, or early embryo, eitheralone or together with a repair template selected from the groupconsisting of SEQ ID NOs:1 to 13. After cleavage and template directedrepair, the CD163 gene can be edited to incorporate the repair templatesequences, and the edited genome of a CD163 gene edited cell cancomprise a sequence selected from the group consisting of SEQ ID NOs: 1to 13.

In one aspect, the present disclosure can include a simple, precise, andreproducible single CD163 loss of function edit in elite pigs such thatthe edit occurs early in the CD163 gene (exons 1-7). In an aspect, a pigcan have a CD163 gene comprising a sequence selected from the groupconsisting of SEQ ID NOs: 1 to 18 and 426 to 505. Also included andprovided for is a pig that can comprise SEQ ID NOs: 1 to 18 and 425 to505 in a genomic region comprising the markers of Table 8. Also includedand provided for is a pig that can comprise SEQ ID NO: 2 in a genomicregion comprising the markers of Table 8. Also included and provided foris a pig that can comprise SEQ ID NO: 426-458 in a genomic regioncomprising the markers of Table 8. Also included and provided for is apig that can comprise SEQ ID NO: 459-504 in a genomic region comprisingthe markers of Table 8. Using porcine embryonic fibroblast cell linesderived from these elite pigs, 250 guide RNA and Cas9 proteincombinations can be tested to identify gene editing pairs thatefficiently cut and generate edits in the CD163 gene. Selected specificguides and endonuclease protein combinations found are listed in Table3, along with the location of the target, their endonuclease activity(average editing frequency), and the position on CD163 of the edit.Greater average editing frequency combinations are preferred becausethey have more effective editing activity. Combinations having anaverage editing frequency greater than 1 are desired. In an aspect,combinations can have an average editing frequency greater than 5. Inanother aspect, combinations can have an average editing frequencygreater than 10. In yet another aspect, combinations can have an averageediting frequency greater than 15. The present disclosure provides for,and includes, combinations that can have an average editing frequencygreater than 20. In an aspect, combinations can have an average editingfrequency greater than 25. In yet another aspect, combinations can havean average editing frequency greater than 30.

As shown in Table 3, the average editing frequency varied greatlybetween target sequences. Moreover, sequences targeting overlappingsequences resulted in different editing frequencies depending on the Casprotein source. For example, SEQ ID NOs: 24 and 25 targeted overlappinglocations and had a 10-fold difference in editing frequency. At thissite, with these sequences, S. pyogenes provided a nearly a 20-foldhigher editing frequency. In contrast, RNPs incorporating SEQ ID NOs: 33and 34 resulted in indistinguishable average editing frequencies betweenthe Cas proteins. While RNPs incorporating a Cas9 nuclease from S.pyogenes can provide superior average editing frequencies, that is notalways the case. As shown for SEQ ID NOs: 60 and 61, RNPs incorporatingthe Cas9 protein from S. thermophilus resulted in superior editingfrequencies (e.g., 3.9 vs. 22.0). Accordingly, the present specificationprovides for efficient guide RNA sequences or efficient targetingsequences for targeting CD163 using the CRISPR/Cas system. In an aspect,RNPs can comprise a sequence selected from the group consisting of thefirst 20 nucleotides of each of SEQ ID NOs: 22 to 271 and 347 to 425.

All position designations herein refer to the Sscrofa11.1 referencegenome (GenBank accession: GCA_000003025.6).

Target site sequences SEQ ID NOs: 22 to 271 and 347 to 425 can beediting target sequences within the porcine CD163 gene. In each targetsite sequence, the first 20 nucleotides can correspond to the guide RNAspacer, and the remaining 3 or 5 nucleotides can be the PAM sequencesfor S. pyogenes or S. thermophilus, respectively. The first 20 bases ofSEQ ID NOs: 22 to 271 and 347 to 425 correspond to the DNA equivalentguide RNA sequences, with the appropriate ribonuclease bases substitutedfor deoxyribonuclease bases, although the DNA bases can be used insteadof RNA bases as guides. Guide RNA sequences can be paired with S.pyogenes or S. thermophilus endonucleases to direct DNA breaks in theporcine genome in either coding or non-coding regions of the CD163 gene,which can result in DNA deletions and/or DNA nucleotide insertions.Depending on the locations of the target sites as disclosed in Table 3,pairs of guide/endonuclease combinations can be selected to deleteregions of the CD163 gene between the pairs of guide/endonucleasecombinations.

Target site sequences SEQ ID NOs: 212 to 271 and 347 to 425 aretargeting sequences that can be used to design guides for deletion ofexon 7 of the CD163 gene. Pairing of intron 6 targeting guides SEQ IDNOs: 212 to 253, and 262 to 271 with intron 7 targeting guides SEQ IDNOs: 254 to 261 using S. pyogenes can cause two double strand breaks onthe CD163 gene, resulting in excision of exon 7. Pairing of targetingguides SEQ ID NOs: 249 and 256 also can result in excision of Exon 7.Pairing of some guides can result in the introduction of a stop codon inExon 7, including SEQ ID NOs: 351 and 365, SEQ ID NOs: 351 and 387, SEQNOs.: 348 and 390, SEQ ID NOs: 348 and 388, SEQ ID NOs: 348 and 498, SEQID NOs: 352 and 365, SEQ ID NOs: 352 and 387, SEQ ID NOs: 352 and 399,SEQ ID NOs: 353 and 365, SEQ ID NOs: 353 and 387, SEQ ID NOs: 353 and399, SEQ ID NOs: 354 and 390, SEQ ID NOs: 354 and 388, SEQ ID NOs: 354and 395, SEQ ID NOs: 358 and 361, SEQ ID NOs: 358 and 362, SEQ ID NOs:358 and 368, SEQ ID NOs: 358 and 384, SEQ ID NOs: 358 and 394, SEQ IDNOs: 358 and 399, SEQ ID NOs: 359 and 390, SEQ ID NOs: 359 and 388, SEQID NOs: 359 and 395, SEQ ID NOs: 360 and 368, SEQ ID NOs: 360 and 384,SEQ ID NOs: 360 and 389, SEQ ID NOs: 360 and 394, SEQ ID NOs: 360 and397, SEQ ID NOs: 361 and 365, SEQ ID NOs: 361 and 387, SEQ ID NOs: 362and 390, SEQ ID NOs: 362 and 388, SEQ ID NOs: 362 and 395, SEQ ID NOs.364 and 365, SEQ ID NOs: 364 and 387, SEQ ID NOs: 364 and 399, SEQ IDNOs: 365 and 368, SEQ ID NOs: 365 and 384, SEQ ID NOs: 365 and 389, SEQID NOs: 365 and 394, SEQ ID NOs: 365 and 397, SEQ ID NOs: 366 and 368,SEQ ID NOs: 366 and 384, SEQ ID NOs: 366 and 389, SEQ ID NOs: 366 and394, or SEQ ID NOs: 366 and 397.

For each targeting sequence, Table 3 lists its SEQ ID No., the speciesof CAS9 nuclease homing arm used, the location of the targeting sequenceon the Sus scrofa genome including the PAM sequence, the editingefficiency as measured by the average fraction of edits for a particularguide in fetal fibroblast cell assays, and the exon on CD163 targeted.

TABLE 3 List of target sequences and editing activities in porcine fetalfibroblasts SEQ Average Position ID edited on NO: Cas9 Nuclease Locationof target fraction CD163 22 S. pyogenes chr5: 63300192-63300214 12.9Exon 1/15 23 S. pyogenes chr5: 63300222-63300244 1.4 Exon 1/15 24 S.pyogenes chr5: 63300236-63300258 5.8 Exon 1/15 25 S. thermophilus chr5:63300236-63300260 0.3 Exon 1/15 26 S. pyogenes chr5: 63300250-633002728.6 Exon 1/15 27 S. pyogenes chr5: 63300251-63300273 0.1 Exon 1/15 28 S.pyogenes chr5: 63300275-63300297 25.7 Exon 1/15 29 S. pyogenes chr5:63300288-63300310 24.9 Exon 1/15 30 S. pyogenes chr5: 63300293-6330031512.8 Exon 1/15 31 S. pyogenes chr5: 63300305-63300327 0.4 Exon 1/15 32S. pyogenes chr5: 63300308-63300330 3.4 Exon 1/15 33 S. thermophiluschr5: 63300308-63300332 3.7 Exon 1/15 34 S. pyogenes chr5:63300327-63300349 5.3 Exon 1/15 35 S. pyogenes chr5: 63300336-633003583.5 Exon 1/15 36 S. pyogenes chr5: 63301950-63301972 14.4 Exon 2/15 37S. pyogenes chr5: 63301951-63301973 16.3 Exon 2/15 38 S. pyogenes chr5:63301962-63301984 13.6 Exon 2/15 39 S. pyogenes chr5: 63301981-633020033.6 Exon 2/15 40 S. pyogenes chr5: 63301995-63302017 21.8 Exon 2/15 41S. pyogenes chr5: 63301997-63302019 8.2 Exon 2/15 42 S. thermophiluschr5: 63301997-63302021 10.1 Exon 2/15 43 S. pyogenes chr5:63303121-63303143 16.4 Exon 3/15 44 S. thermophilus chr5:63303121-63303145 16.1 Exon 3/15 45 S. pyogenes chr5: 63303129-6330315131.4 Exon 3/15 46 S. pyogenes chr5: 63303136-63303158 22.8 Exon 3/15 47S. pyogenes chr5: 63303137-63303159 42.9 Exon 3/15 48 S. thermophiluschr5: 63303137-63303161 10.7 Exon 3/15 49 S. pyogenes chr5:63303140-63303162 36.7 Exon 3/15 50 S. thermophilus chr5:63303140-63303164 32.6 Exon 3/15 51 S. pyogenes chr5: 63303158-6330318021.1 Exon 3/15 52 S. pyogenes chr5: 63303166-63303188 23.6 Exon 3/15 53S. thermophilus chr5: 63303166-63303190 22.1 Exon 3/15 54 S. pyogeneschr5: 63303169-63303191 40.0 Exon 3/15 55 S. thermophilus chr5:63303169-63303193 19.5 Exon 3/15 56 S. pyogenes chr5: 63303181-633032039.9 Exon 3/15 57 S. thermophilus chr5: 63303181-63303205 10.4 Exon 3/1558 S. pyogenes chr5: 63303184-63303206 20.6 Exon 3/15 59 S. thermophiluschr5: 63303184-63303208 2.8 Exon 3/15 60 S. pyogenes chr5:63303189-63303211 3.9 Exon 3/15 61 S. thermophilus chr5:63303189-63303213 22.0 Exon 3/15 62 S. pyogenes chr5: 63303190-6330321218.1 Exon 3/15 63 S. pyogenes chr5: 63303191-63303213 23.5 Exon 3/15 64S. pyogenes chr5: 63303209-63303231 23.6 Exon 3/15 65 S. pyogenes chr5:63303213-63303235 4.1 Exon 3/15 66 S. pyogenes chr5: 63303214-6330323616.2 Exon 3/15 67 S. pyogenes chr5: 63303220-63303242 14.6 Exon 3/15 68S. pyogenes chr5: 63303226-63303248 15.4 Exon 3/15 69 S. pyogenes chr5:63303243-63303265 21.6 Exon 3/15 70 S. pyogenes chr5: 63303250-633032721.5 Exon 3/15 71 S. pyogenes chr5: 63303251-63303273 30.3 Exon 3/15 72S. pyogenes chr5: 63303278-63303300 20.1 Exon 3/15 73 S. pyogenes chr5:63303278-63303300 1.5 Exon 3/15 74 S. pyogenes chr5: 63303282-6330330423.9 Exon 3/15 75 S. pyogenes chr5: 63303283-63303305 20.8 Exon 3/15 76S. pyogenes chr5: 63303294-63303316 33.8 Exon 3/15 77 S. pyogenes chr5:63303299-63303321 8.2 Exon 3/15 78 S. pyogenes chr5: 63303305-633033276.1 Exon 3/15 79 S. pyogenes chr5: 63303315-63303337 25.2 Exon 3/15 80S. pyogenes chr5: 63303319-63303341 28.7 Exon 3/15 81 S. pyogenes chr5:63303338-63303360 4.5 Exon 3/15 82 S. pyogenes chr5: 63303339-633033612.8 Exon 3/15 83 S. pyogenes chr5: 63303357-63303379 18.3 Exon 3/15 84S. pyogenes chr5: 63303358-63303380 26.6 Exon 3/15 85 S. pyogenes chr5:63303374-63303396 16.1 Exon 3/15 86 S. pyogenes chr5: 63303378-6330340035.4 Exon 3/15 87 S. thermophilus chr5: 63303378-63303402 40.0 Exon 3/1588 S. pyogenes chr5: 63303379-63303401 24.4 Exon 3/15 89 S. pyogeneschr5: 63303380-63303402 25.6 Exon 3/15 90 S. pyogenes chr5:63303406-63303428 28.1 Exon 3/15 91 S. pyogenes chr5: 63303413-6330343513.7 Exon 3/15 92 S. thermophilus chr5: 63303413-63303437 19.0 Exon 3/1593 S. thermophilus chr5: 63303419-63303443 15.2 Exon 3/15 94 S. pyogeneschr5: 63303421-63303443 16.5 Exon 3/15 95 S. pyogenes chr5:63303428-63303450 35.8 Exon 3/15 96 S. pyogenes chr5: 63303441-633034633.4 Exon 3/15 97 S. pyogenes chr5: 63306087-63306109 19.8 Exon 4/15 98S. pyogenes chr5: 63306091-63306113 4.5 Exon 4/15 99 S. thermophiluschr5: 63306091-63306115 3.1 Exon 4/15 100 S. pyogenes chr5:63306098-63306120 29.9 Exon 4/15 101 S. thermophilus chr5:63306098-63306122 31.5 Exon 4/15 102 S. pyogenes chr5: 63306101-6330612329.2 Exon 4/15 103 S. pyogenes chr5: 63306108-63306130 6.9 Exon 4/15 104S. thermophilus chr5: 63306108-63306132 14.7 Exon 4/15 105 S. pyogeneschr5: 63306116-63306138 21.6 Exon 4/15 106 S. pyogenes chr5:63306127-63306149 26.4 Exon 4/15 107 S. pyogenes chr5: 63306140-633061626.9 Exon 4/15 108 S. pyogenes chr5: 63306144-63306166 35.2 Exon 4/15 109S. thermophilus chr5: 63306144-63306168 3.3 Exon 4/15 110 S. pyogeneschr5: 63306147-63306169 8.8 Exon 4/15 111 S. thermophilus chr5:63306147-63306171 3.2 Exon 4/15 112 S. pyogenes chr5: 63306148-633061705.2 Exon 4/15 113 S. pyogenes chr5: 63306149-63306171 28.0 Exon 4/15 114S. pyogenes chr5: 63306193-63306215 9.3 Exon 4/15 115 S. pyogenes chr5:63306236-63306258 14.2 Exon 4/15 116 S. pyogenes chr5: 63306251-6330627326.3 Exon 4/15 117 S. thermophilus chr5: 63306251-63306275 4.4 Exon 4/15118 S. pyogenes chr5: 63306257-63306279 1.3 Exon 4/15 119 S. pyogeneschr5: 63306263-63306285 7.8 Exon 4/15 120 S. pyogenes chr5:63306273-63306295 35.0 Exon 4/15 121 S. pyogenes chr5: 63306287-6330630913.4 Exon 4/15 122 S. pyogenes chr5: 63306296-63306318 2.0 Exon 4/15 123S. pyogenes chr5: 63306315-63306337 31.2 Exon 4/15 124 S. pyogenes chr5:63306332-63306354 10.1 Exon 4/15 125 S. pyogenes chr5: 63306336-6330635831.1 Exon 4/15 126 S. thermophilus chr5: 63306336-63306360 52.8 Exon4/15 127 S. pyogenes chr5: 63306337-63306359 30.3 Exon 4/15 128 S.pyogenes chr5: 63306338-63306360 43.2 Exon 4/15 129 S. pyogenes chr5:63306364-63306386 2.5 Exon 4/15 130 S. pyogenes chr5: 63306371-633063930.2 Exon 4/15 131 S. thermophilus chr5: 63306371-63306395 0.3 Exon 4/15132 S. pyogenes chr5: 63309028-63309050 4.5 Exon 5/15 133 S. pyogeneschr5: 63309034-63309056 5.1 Exon 5/15 134 S. pyogenes chr5:63309035-63309057 41.8 Exon 5/15 135 S. thermophilus chr5:63309035-63309059 21.9 Exon 5/15 136 S. pyogenes chr5: 63309053-6330907515.1 Exon 5/15 137 S. pyogenes chr5: 63309061-63309083 0.2 Exon 5/15 138S. pyogenes chr5: 63309077-63309099 15.7 Exon 5/15 139 S. thermophiluschr5: 63309077-63309101 11.2 Exon 5/15 140 S. pyogenes chr5:63309084-63309106 10.1 Exon 5/15 141 S. thermophilus chr5:63309084-63309108 23.8 Exon 5/15 142 S. pyogenes chr5: 63309085-6330910711.3 Exon 5/15 143 S. pyogenes chr5: 63309086-63309108 23.4 Exon 5/15144 S. pyogenes chr5: 63309094-63309116 13.8 Exon 5/15 145 S. pyogeneschr5: 63309104-63309126 7.8 Exon 5/15 146 S. pyogenes chr5:63309108-63309130 1.4 Exon 5/15 147 S. pyogenes chr5: 63309109-633091317.0 Exon 5/15 148 S. pyogenes chr5: 63309130-63309152 24.8 Exon 5/15 149S. pyogenes chr5: 63309144-63309166 20.8 Exon 5/15 150 S. pyogenes chr5:63309145-63309167 0.1 Exon 5/15 151 S. pyogenes chr5: 63309146-6330916837.4 Exon 5/15 152 S. pyogenes chr5: 63309173-63309195 2.5 Exon 5/15 153S. pyogenes chr5: 63309173-63309195 2.3 Exon 5/15 154 S. pyogenes chr5:63309189-63309211 11.5 Exon 5/15 155 S. pyogenes chr5: 63309193-633092152.7 Exon 5/15 156 S. pyogenes chr5: 63309194-63309216 10.4 Exon 5/15 157S. pyogenes chr5: 63309200-63309222 3.8 Exon 5/15 158 S. thermophiluschr5: 63309205-63309229 19.0 Exon 5/15 159 S. pyogenes chr5:63309207-63309229 21.0 Exon 5/15 160 S. pyogenes chr5: 63309210-6330923219.0 Exon 5/15 161 S. pyogenes chr5: 63309233-63309255 8.3 Exon 5/15 162S. pyogenes chr5: 63309250-63309272 13.6 Exon 5/15 163 S. pyogenes chr5:63309252-63309274 23.0 Exon 5/15 164 S. pyogenes chr5: 63309273-633092952.3 Exon 5/15 165 S. thermophilus chr5: 63309273-63309297 34.4 Exon 5/15166 S. pyogenes chr5: 63309274-63309296 15.1 Exon 5/15 167 S. pyogeneschr5: 63309275-63309297 19.4 Exon 5/15 168 S. thermophilus chr5:63309284-63309308 14.4 Exon 5/15 169 S. pyogenes chr5: 63309286-633093087.0 Exon 5/15 170 S. pyogenes chr5: 63309308-63309330 0.5 Exon 5/15 171S. thermophilus chr5: 63309308-63309332 2.4 Exon 5/15 172 S. pyogeneschr5: 63309323-63309345 16.0 Exon 5/15 173 S. pyogenes chr5:63309841-63309863 0.1 Exon 6/15 174 S. pyogenes chr5: 63309857-633098798.4 Exon 6/15 175 S. thermophilus chr5: 63309857-63309881 8.0 Exon 6/15176 S. pyogenes chr5: 63309860-63309882 40.3 Exon 6/15 177 S.thermophilus chr5: 63309860-63309884 32.3 Exon 6/15 178 S. pyogeneschr5: 63309863-63309885 37.2 Exon 6/15 179 S. pyogenes chr5:63309886-63309908 4.7 Exon 6/15 180 S. thermophilus chr5:63309886-63309910 3.5 Exon 6/15 181 S. pyogenes chr5: 63309889-6330991138.1 Exon 6/15 182 S. pyogenes chr5: 63309889-63309911 34.1 Exon 6/15183 S. thermophilus chr5: 63309889-63309913 17.5 Exon 6/15 184 S.pyogenes chr5: 63309892-63309914 4.9 Exon 6/15 185 S. pyogenes chr5:63309907-63309929 13.5 Exon 6/15 186 S. pyogenes chr5: 63309911-633099335.5 Exon 6/15 187 S. pyogenes chr5: 63309933-63309955 0.7 Exon 6/15 188S. thermophilus chr5: 63309933-63309957 10.0 Exon 6/15 189 S. pyogeneschr5: 63309934-63309956 6.4 Exon 6/15 190 S. pyogenes chr5:63309935-63309957 18.7 Exon 6/15 191 S. pyogenes chr5: 63309955-6330997712.0 Exon 6/15 192 S. pyogenes chr5: 63309963-63309985 5.6 Exon 6/15 193S. pyogenes chr5: 63309970-63309992 0.5 Exon 6/15 194 S. pyogenes chr5:63309971-63309993 12.5 Exon 6/15 195 S. pyogenes chr5: 63309977-633099995.3 Exon 6/15 196 S. pyogenes chr5: 63310021-63310043 2.2 Exon 6/15 197S. pyogenes chr5: 63310035-63310057 10.3 Exon 6/15 198 S. pyogenes chr5:63310038-63310060 0.2 Exon 6/15 199 S. pyogenes chr5: 63310058-633100808.2 Exon 6/15 200 S. pyogenes chr5: 63310077-63310099 6.2 Exon 6/15 201S. pyogenes chr5: 63310078-63310100 8.6 Exon 6/15 202 S. pyogenes chr5:63310092-63310114 2.7 Exon 6/15 203 S. pyogenes chr5: 63310098-6331012011.1 Exon 6/15 204 S. thermophilus chr5: 63310098-63310122 8.0 Exon 6/15205 S. pyogenes chr5: 63310099-63310121 12.2 Exon 6/15 206 S. pyogeneschr5: 63310100-63310122 21.1 Exon 6/15 207 S. thermophilus chr5:63310100-63310124 0.1 Exon 6/15 208 S. pyogenes chr5: 63310103-633101253.3 Exon 6/15 209 S. pyogenes chr5: 63310152-63310174 4.8 Exon 6/15 210S. pyogenes chr5: 63323061-63323083 32.8 Exon 7/15 211 S. pyogenes chr5:63323147-63323169 17.0 Exon 7/15 212 S. pyogenes chr5: 63322548-6332257013.6 Intron 6 213 S. pyogenes chr5: 63322549-63322571 1.7 Intron 6 214S. pyogenes chr5: 63322566-63322588 15.6 Intron 6 215 S. pyogenes chr5:63322594-63322616 0.0 Intron 6 216 S. pyogenes chr5: 63322597-633226190.0 Intron 6 217 S. pyogenes chr5: 63322646-63322668 21.1 Intron 6 218S. pyogenes chr5: 63322647-63322669 12.6 Intron 6 219 S. pyogenes chr5:63322681-63322703 44.3 Intron 6 220 S. pyogenes chr5: 63322683-633227053.9 Intron 6 221 S. pyogenes chr5: 63322693-63322715 33.9 Intron 6 222S. pyogenes chr5: 63322694-63322716 26.1 Intron 6 223 S. pyogenes chr5:63322714-63322736 5.7 Intron 6 224 S. pyogenes chr5: 63322731-6332275342.8 Intron 6 225 S. pyogenes chr5: 63322756-63322778 5.8 Intron 6 226S. pyogenes chr5: 63322757-63322779 21.2 Intron 6 227 S. pyogenes chr5:63322770-63322792 36.1 Intron 6 228 S. pyogenes chr5: 63322799-6332282129.9 Intron 6 229 S. pyogenes chr5: 63322800-63322822 43.2 Intron 6 230S. pyogenes chr5: 63322809-63322831 33.3 Intron 6 231 S. pyogenes chr5:63322810-63322832 46.8 Intron 6 232 S. pyogenes chr5: 63322834-633228567.8 Intron 6 233 S. pyogenes chr5: 63322835-63322857 18.3 Intron 6 234S. pyogenes chr5: 63322839-63322861 18.8 Intron 6 235 S. pyogenes chr5:63322839-63322861 13.2 Intron 6 236 S. pyogenes chr5: 63322840-633228622.8 Intron 6 237 S. pyogenes chr5: 63322845-63322867 55.3 Intron 6 238S. pyogenes chr5: 63322848-63322870 27.2 Intron 6 239 S. pyogenes chr5:63322852-63322874 24.4 Intron 6 240 S. pyogenes chr5: 63322859-6332288122.2 Intron 6 241 S. pyogenes chr5: 63322875-63322897 25.3 Intron 6 242S. pyogenes chr5: 63322887-63322909 4.2 Intron 6 243 S. pyogenes chr5:63322888-63322910 3.5 Intron 6 244 S. pyogenes chr5: 63322891-6332291344.3 Intron 6 245 S. pyogenes chr5: 63322900-63322922 58.2 Intron 6 246S. pyogenes chr5: 63322906-63322928 3.7 Intron 6 247 S. pyogenes chr5:63322926-63322948 38.1 Intron 6 248 S. pyogenes chr5: 63322927-633229490.5 Intron 6 249 S. pyogenes chr5: 63322947-63322969 31.7 Intron 6 250S. pyogenes chr5: 63322957-63322979 50.0 Intron 6 251 S. pyogenes chr5:63322957-63322979 11.0 Intron 6 252 S. pyogenes chr5: 63322991-633230134.5 Intron 6 253 S. pyogenes chr5: 63322992-63323014 0.5 Intron 6 254 S.pyogenes chr5: 63323338-63323360 19.8 Intron 7 255 S. pyogenes chr5:63323339-63323361 8.1 Intron 7 256 S. pyogenes chr5: 63323361-6332338321.1 Intron 7 257 S. pyogenes chr5: 63323362-63323384 30.4 Intron 7 258S. pyogenes chr5: 63323362-63323384 5.6 Intron 7 259 S. pyogenes chr5:63323363-63323385 2.8 Intron 7 260 S. pyogenes chr5: 63323367-6332338923.2 Intron 7 261 S. pyogenes chr5: 63323368-63323390 26.2 Intron 7 262S. thermophilus chr5: 63322644-63322668 Activity Intron 6 not tested 263S. thermophilus chr5: 63322647-63322671 Activity Intron 6 not tested 264S. thermophilus chr5: 63322678-63322702 Activity Intron 6 not tested 265S. thermophilus chr5: 63322681-63322705 Activity Intron 6 not tested 266S. thermophilus chr5: 63322755-63322779 Activity Intron 6 not tested 267S. thermophilus chr5: 63322807-63322831 Activity Intron 6 not tested 268S. thermophilus chr5: 63322845-63322869 Activity Intron 6 not tested 269S. thermophilus chr5: 63322850-63322874 Activity Intron 6 not tested 270S. thermophilus chr5: 63322955-63322979 Activity Intron 6 not tested 271S. thermophilus chr5: 63322989-63323013 Activity Intron 6 not tested 347S. pyogenes chr5: 63323002-63323024 2.1 Exon 7 348 S. pyogenes chr5:63323011-63323033 31.9 Exon 7 349 S. pyogenes chr5: 63323015-633230377.7 Exon 7 350 S. pyogenes chr5: 63323017-63323039 56.5 Exon 7 351 S.pyogenes chr5: 63323018-63323040 74.3 Exon 7 352 S. pyogenes chr5:63323019-63323041 52.3 Exon 7 353 S. pyogenes chr5: 63323022-6332304444.0 Exon 7 354 S. pyogenes chr5: 63323023-63323045 61.6 Exon 7 355 S.pyogenes chr5: 63323024-63323046 49.4 Exon 7 356 S. pyogenes chr5:63323028-63323050 3.1 Exon 7 357 S. pyogenes chr5: 63323029-63323051 0.8Exon 7 358 S. pyogenes chr5: 63323040-63323062 42.4 Exon 7 359 S.pyogenes chr5: 63323052-63323074 66.7 Exon 7 360 S. pyogenes chr5:63323053-63323075 13.4 Exon 7 361 S. pyogenes chr5: 63323061-6332308377.9 Exon 7 362 S. pyogenes chr5: 63323071-63323093 60.8 Exon 7 363 S.pyogenes chr5: 63323072-63323094 70.6 Exon 7 364 S. pyogenes chr5:63323073-63323095 75.2 Exon 7 365 S. pyogenes chr5: 63323098-6332312064.4 Exon 7 366 S. pyogenes chr5: 63323102-63323124 55.0 Exon 7 367 S.pyogenes chr5: 63323105-63323127 51.2 Exon 7 368 S. pyogenes chr5:63323108-63323130 58.3 Exon 7 369 S. pyogenes chr5: 63323125-6332314714.5 Exon 7 370 S. pyogenes chr5: 63323126-63323148 22.6 Exon 7 371 S.pyogenes chr5: 63323131-63323153 49.9 Exon 7 372 S. pyogenes chr5:63323139-63323161 68.9 Exon 7 373 S. pyogenes chr5: 63323147-6332316957.4 Exon 7 374 S. pyogenes chr5: 63323159-63323181 12.9 Exon 7 375 S.pyogenes chr5: 63323160-63323182 19.7 Exon 7 376 S. pyogenes chr5:63323161-63323183 33.6 Exon 7 377 S. pyogenes chr5: 63323162-6332318465.3 Exon 7 378 S. pyogenes chr5: 63323163-63323185 51.4 Exon 7 379 S.pyogenes chr5: 63323173-63323195 57.6 Exon 7 380 S. pyogenes chr5:63323174-63323196 52.1 Exon 7 381 S. pyogenes chr5: 63323175-6332319748.2 Exon 7 382 S. pyogenes chr5: 63323177-63323199 33.7 Exon 7 383 S.pyogenes chr5: 63323181-63323203 18.3 Exon 7 384 S. pyogenes chr5:63323187-63323209 57.0 Exon 7 385 S. pyogenes chr5: 63323197-6332321933.3 Exon 7 386 S. pyogenes chr5: 63323198-63323220 58.3 Exon 7 387 S.pyogenes chr5: 63323219-63323241 20.7 Exon 7 388 S. pyogenes chr5:63323220-63323242 42.2 Exon 7 389 S. pyogenes chr5: 63323221-6332324343.4 Exon 7 390 S. pyogenes chr5: 63323231-63323253 21.8 Exon 7 391 S.pyogenes chr5: 63323251-63323273 46.8 Exon 7 392 S. pyogenes chr5:63323252-63323274 42.6 Exon 7 393 S. pyogenes chr5: 63323255-6332327722.8 Exon 7 394 S. pyogenes chr5: 63323267-63323289 85.4 Exon 7 395 S.pyogenes chr5: 63323268-63323290 41.1 Exon 7 396 S. pyogenes chr5:63323268-63323290 53.6 Exon 7 397 S. pyogenes chr5: 63323269-6332329132.1 Exon 7 398 S. pyogenes chr5: 63323277-63323299 16.6 Exon 7 399 S.pyogenes chr5: 63323278-63323300 39.0 Exon 7 400 S. pyogenes chr5:63323279-63323301 11.7 Exon 7 401 S. pyogenes chr5: 63323282-6332330448.9 Exon 7 402 S. pyogenes chr5: 63323283-63323305 n/a Exon 7 403 S.pyogenes chr5: 63323287-63323309 11.6 Exon 7 404 S. pyogenes chr5:63323288-63323310 69.3 Exon 7 405 S. pyogenes chr5: 63323295-6332331746.0 Exon 7 406 S. pyogenes chr5: 63323300-63323322 54.2 Exon 7 407 S.thermophilus chr5: 63323019-63323043 25.5 Exon 7 408 S. thermophiluschr5: 63323022-63323046 24.7 Exon 7 409 S. thermophilus chr5:63323061-63323085 37.8 Exon 7 410 S. thermophilus chr5:63323071-63323095 30.4 Exon 7 411 S. thermophilus chr5:63323096-63323120 33.4 Exon 7 412 S. thermophilus chr5:63323102-63323126 19.8 Exon 7 413 S. thermophilus chr5:63323105-63323129 0.6 Exon 7 414 S. thermophilus chr5: 63323159-6332318314.8 Exon 7 415 S. thermophilus chr5: 63323160-63323184 7.8 Exon 7 416S. thermophilus chr5: 63323161-63323185 3.6 Exon 7 417 S. thermophiluschr5: 63323163-63323187 7.9 Exon 7 418 S. thermophilus chr5:63323175-63323199 33.8 Exon 7 419 S. thermophilus chr5:63323175-63323199 11.5 Exon 7 420 S. thermophilus chr5:63323219-63323243 8.1 Exon 7 421 S. thermophilus chr5: 63323253-6332327732.6 Exon 7 422 S. thermophilus chr5: 63323275-63323299 0.2 Exon 7 423S. thermophilus chr5: 63323277-63323301 12.3 Exon 7 424 S. thermophiluschr5: 63323280-63323304 26.0 Exon 7 425 S. thermophilus chr5:63323295-63323319 30.5 Exon 7

Efficient guideRNA-Cas9 pairs which cut sites early in the CD163 gene(exons 1-4) were selected and further screened for potential off-targetbinding within the pig genome.

In some instances, the use of guide RNA and endonuclease has beenobserved to result in cleavage of DNA at unintended locations in thegenome. Because the repair process for dsDNA breaks can be random,off-target cleavage events can result in undesirable changes to codingor regulatory regions in the genome. Therefore, in instances where anumber of single or paired guides can perform an intended edit withsimilar editing frequencies, it is advantageous to consider off-targetcleavage in choosing a guide or guide pair for editing experiments.

A number of computational and biochemical approaches for elucidatingoff-targets have been developed. Computational approaches can include,but are not limited to, Cas-OFFinder (Bae, S., et al., Bioinformatics,2014, 30, 1473-1475.), CRISPR-offender (Zhao, C., et al., Int. J. Biol.Sci., 2017, 13, 1470-1478), and CRISPR-OFF (Alkan, F., et al., GenomeBiol., 2018, 19, 177). Other computational approaches are readilyavailable to those skilled in the art. Biochemical approaches caninclude, but are not limited to, GUIDE-Seq (Tsai, S. Q., et al., NatBiotechnol. 2015, 33, 187-197), SITE-SEQ® (Cameron, P., et al., NatMethods 2017, 14, 600-606), and CIRCLE-seq (Tsai, S. Q., et al., Nat.Methods, 2017, 14, 607-614). Other biochemical approaches are readilyavailable to those skilled in the art. While computational methods arerelatively fast and inexpensive compared to biochemical ones,biochemical approaches have been shown to be superior in identifyingvalidated off-target edits.

A subset of guideRNAs that have demonstrated a high frequency ofintended edits in porcine fetal fibroblasts, as described above, wereassayed for specificity using SITE-SEQ®. Using naked gDNA and RNPediting reagents in vitro, SITE-SEQ® can provide a list of potentialcleavage sites. Off-target cleavage in a cellular environment can bemore complicated than that simulated biochemically in vitro. Factorssuch as effective RNP concentration and target availability due tochromatin state, among other factors, can contribute to the number ofoff-target edits realized in porcine cells or in edited pigs.Fortunately, biochemical methods such as SITE-SEQ® can provideresearchers a list of sites to interrogate. These sites can beinterrogated by methods that include, but are not limited to, TOPOcloning and sequencing of TOPO clones, ILLUMINA® amplicon sequencing,Nanopore sequencing, other NGS sequencing methods, and sequence capture(Gnirke, A., et al., Nat. Biotechnol., 2009, 27, 182-189).

Screening was performed for biochemically-identified off-target sites inedited porcine fibroblasts and subsequently in injected embryos. Guideswith validated off-target edits were de-prioritized for use ingenerating edited pigs. Animals generated using guides with knownoff-targets can be interrogated for the presence of off-target editsusing the strategies outlined above. Animals that do contain off-targetedits can either be removed from the breeding program or the off-targetedits can be removed via breeding. This screening can includebioinformatic methods, such as BLAST® searching, to identify sequencesin the Sus scrofa genome that contain 1-5 mismatches with the guideRNA,that could therefore allow for off-target binding. The number ofpotential off-target binding sites in the genome when allowing thesemismatches in the computer algorithm is detailed below in Table 4.

TABLE 4 Mismatch Detection SEQ ID MISMATCHES ≤ 5 _ LOCATION-Spy SEQ SEQID Stherm SITE coordinates ID Spy Stherm Spy C3 1 chr5:63300236-63300258 19 20 920 142 2 chr5: 63300308-63300330 32 33 625 51 3chr5: 63301997-63302019 41 42 506 37 4 chr5: 63303121-63303143 43 441625 201 5 chr5: 63303137-63303159 47 48 474 32 6 chr5:63303140-63303162 49 50 548 49 7 chr5: 63303166-63303188 52 53 692 92 8chr5: 63303169-63303191 54 55 782 94 9 chr5: 63303181-63303203 56 571346 200 10 chr5: 63303184-63303206 58 59 1873 382 11 chr5:63303189-63303211 60 61 1642 240 12 chr5: 63303378-63303400 86 87 588 5313 chr5: 63303413-63303435 91 92 545 50 14 chr5: 63306108-63306130 103104 480 49 15 chr5: 63306144-63306166 108 109 344 21 16 chr5:63306147-63306169 110 111 451 44 17 chr5: 63306251-63306273 116 117 134365 18 chr5: 63306336-63306358 125 126 718 66 19 chr5: 63306371-63306393130 131 1310 131 20 chr5: 63309035-63309057 134 135 690 83 21 chr5:63309077-63309099 138 139 949 95 22 chr5: 63309084-63309106 140 141 123499 23 chr5: 63309273-63309295 164 165 418 47 24 chr5: 63309308-63309330170 171 747 95 25 chr5: 63309857-63309879 174 175 795 47 26 chr5:63309860-63309882 176 177 736 94 27 chr5: 63309886-63309908 179 180 1829124 28 chr5: 63309889-63309911 182 183 1575 94 29 chr5:63309933-63309955 187 188 871 95 30 chr5: 63310098-63310120 203 204 53373 31 chr5: 63310100-63310122 206 207 537 53

One of the factors that contributes to cutting at these off-target sitescan be whether the gene editing components are delivered as a DNA vectoror as an RNA-protein complex; sites that may have as few as 1 or 2 basemismatches can be faithfully discriminated from the intended target sitewhen the Cas9 and guide RNA can be delivered as a single guideribonucleotide protein complex (sgRNP) rather than delivered on a DNAvector.

A second consideration to maximize specificity of a Cas9 guide-RNAreagent can be to prescreen guide RNA-protein pairs using in vitrobiochemical methods. Several laboratories have published methods toscreen for off-target edit sites. These biochemical approaches canidentify potential off-target Cas9 cleavage sites in purified genomicDNA. Using these assays, genomic DNA can be digested with a range ofsgRNP concentrations, from limiting to saturating, thus permitting therecovery of both high- and low-cleavage-sensitivity off-target sites.See, e.g., Cameron et al., “SITE-SEQ®: A Genome-wide Method to MeasureCas9 Cleavage,” Protocol Exchange (2017).

The off-target sites identified by off-target screening can be used toguide careful and comprehensive examination of possible off-target sitesin cells, measuring both editing frequency and functional cellularconsequence. Several selected guide RNA-Cas protein pairs can bescreened, and the screening can demonstrate: 1) efficient cutting nearerto the 5′ end of the CD163 gene, and 2) few mismatch sequences asdetermined by bioinformatic methods.

Using this guide selection criteria, several guide RNA-Cas protein pairscan be selected for cutting activity in porcine parthenotes. Porcineoocytes can be doubled using an electric current, injected with guideRNA-Cas protein, allowed to develop for 7 days, then harvested and DNAsequenced across the intended target site. Several guide RNA-Cas proteinpairs can be identified to confer edits at a high frequency inparthenotes and are selected for further development.

The present specification provides for, and includes, methods foridentifying and selecting optimal target sites for CRISPR/Cas mediatedcleavage and gene editing. In an aspect, the method can compriseidentifying a target region of a genome for editing; identifying all 20nucleotide sequences in the target region; performing a bioinformaticscreen to identify and remove sequences that match non-target sites inthe pig genome that contain 1 to 4 mismatches and have a suitable PAMsequence based on the Cas protein; preparing CRISPR/Cas RNP complexescomprising a guide RNA backbone and target sequences, introducing theCRISPR/Cas RNP complexes into a porcine cell in culture, and determiningthe average editing frequency of the guide RNA/Cas combination. In anaspect, determining the average editing frequency of the guide RNA/Cascombination can comprise amplifying, by PCR, a region surrounding thetarget site, performing amplicon deep sequencing, and comparing it tountreated cells. In an aspect, the method provides for selectingpreferred target sequences that can have an average editing frequency ofat least 15. Also provided for are methods for identifying and selectingoptimal target sites for CRISPR/Cas mediated cleavage and gene editingwherein the guide RNA and Cas proteins can be provided as part of anexpression vector or vectors. Suitable cells are known to persons ofskill in the art and can include, but are not limited to, primary fetalfibroblasts of an intended porcine breeding line.

Multiple repair templates can be designed to generate in-frame stopcodons. The repair templates listed, when used with appropriate guideRNAs in an endonuclease system, can generate an in-frame terminationcodon (TAA, TGA, TAG) after repair. Different lengths of repairtemplates can be used, having sequence identity on either side of theedit site. In an aspect, a repair template can share at least 15nucleotides on either side of the CRISPR/Cas endonuclease cleavage site(e.g., 15-15 nucleotides). In other aspects, repair templates can sharemore than 100 nucleotides on either side of the edit site(e.g., >100˜>100). Repair templates can be single, double, or staggeredstrands of complementary DNA using overhangs on the ends. In addition,these templates are not limited to DNA but also could be RNA or modifiednucleotides (inosine, for example) or a mixture of these bases and have5′ and or 3′ ends protected from degradation. Because there areexonucleases that cleave nucleotides from the ends, protecting the endswith modified bases can prevent exonuclease digestion in the cell ofboth DNA and RNA. Not to be limited by theory, increasing the length ofthe region of identity on both sides of the edit site can increase theefficiency and specificity of the repair process (e.g., homology arms).Included and provided for by the present specification are repairtemplates that can have 100% identity over at least 25 nucleotides onboth sides of the edit site. This core identity region ensures that thedesired edit (for example, deletions for SEQ ID NOs: 1 to 13) isaccurate and efficient. In an aspect, the core identity region can be atleast 40 nucleotides flanking the edit site. In yet another aspect, thecore identity region can be at least 50 nucleotides on both sides of theedit site. In an aspect, the flanking core region can comprise 60nucleotides of 100% identity. Also included in an aspect are repairtemplates that can have 70 identical nucleotides to the chromosomalregion flanking the edit site. In an aspect, the core identity regioncan comprise 75 nucleotides of identity to the chromosomal regionflanking the edit site. In an aspect, the core identity region cancomprise 25 to 40 flanking nucleotides. In a further aspect, the coreidentity region can comprise 40 to 75 flanking nucleotides.

In some aspects, the core identity region can be further flanked byadditional regions of homology to the target site (the “flankinghomology regions”). As provided herein, in an aspect, the flankinghomology regions can comprise 100% identity to the target site. Asexamples, SEQ ID NOs: 1 to 13 shared 100% homology to the CD163 regionon either side of the targeted edit site as found in lines 2, 3, 15, 19,27, 62, or 65 (e.g., 100% homology in the core region and the flankinghomology regions). Also included are repair templates that can compriseSEQ ID NOs: 1 to 13, wherein the core identity region can comprise 100%identity to 25 nucleotides of the genome on either side of the desirededit (e.g., bases 50 to 100) and can be flanked by at least 80% identityto the genome on either side of the core region (e.g., nucleotides 1 to49 and 101 to 150 of SEQ ID NOs: 1 to 18). In another aspect, the coreregion can have 100% homology and the flanking homology regions canshare 85% homology. In a further aspect, the core region can have 100%homology and the flanking homology regions can share 90% homology. Inyet another aspect, the core region can have 100% homology and theflanking homology regions can share 95% homology. Also included in anaspect are repair templates that can have a core region that has 100%homology and the flanking homology regions share 97% homology. In someaspects, the flanking homology regions can share 99% homology. Not to belimited by theory, it is thought that increasing the length of theflanking homology regions can introduce the desired CD163 edits intorelated animals without further modifying the genome. That is, specificrepair sequences incorporating polymorphisms or changes in the CD163genome of other pigs are specifically included and provided for.

The present specification includes, and provides for, flanking homologyregions that can have greater than 50 nucleotides on each side of theedit site. In an aspect, the flanking homology regions can have greaterthan 75 nucleotides on each side of the edit site. In an aspect, theflanking homology regions can have greater than 100 nucleotides on eachside of the edit site. Also included are flanking homology regions thatcan have greater than 200 nucleotides on each side of the edit site. Inaspects, the flanking homology regions can have between 30 and 1000nucleotides on each side of the edit site.

The present specification includes, and provides for, additionalmodifications at the 5′ and 3′ ends of repair template. Repair templatesare not limited to DNA but also could be RNA or modified nucleotides(inosine, for example) or a mixture of these bases and can have 5′ andor 3′ ends protected from degradation. Because there are exonucleasesthat cleave nucleotides from the ends, protecting the ends with modifiedbases can prevent exonuclease digestion in the cell of both DNA and RNA.Accordingly, as provided herein, the 5′ and 3′ ends of a repair templatecomprising a flanking homology region can be modified to preventdegradation.

The present specification provides for, and includes, ribonucleoprotein(RNP) complexes that can comprise a guide polynucleotide and a Casprotein. In an aspect, the Cas protein can be a S. thermophilus Cas9. Inanother aspect, the Cas protein can be Cas9 from S. pyogenes. Othersuitable Cas proteins are known in the art and exemplary Cas systems aredescribed above in Table 1. The selection of suitable Cas proteinsdepends on the required PAM sequence combination, and methods ofidentifying Cas proteins and modifying Pam sequences are known.Similarly, Cas systems differ in their requirements for guide RNAbackbone sequences (e.g., tracrRNA sequences). In an aspect, the RNPcomplex can comprise a Cas protein and a guide nucleotide having atleast 98% sequence identity to an RNA sequence selected from the groupconsisting of the first 20 nucleotides of each of SEQ ID NOs: 22 to 271and 347 to 425. In another aspect, the RNP complex can comprise a guideRNA comprising a sequence selected from the group consisting of thefirst 20 nucleotides of each of SEQ ID NOs: 22 to 271 and 347 to 425. Inan aspect, the RNP complex can comprise a guide polynucleotide having99% identity to a sequence selected from the group consisting of thefirst 20 nucleotides of each of SEQ ID NOs: 22 to 271 and 347 to 425. Inan aspect, the guide polynucleotide of the RNP complex can comprise asequence selected from the group consisting of the first 20 nucleotidesof each of SEQ ID NOs: 22 to 271 and 347 to 425. As provided herein, theRNP complex can comprise the sequences of any one of SEQ ID NOs: 22 to271 and 347 to 425 combined with an RNA backbone as part of an sgRNA. Inan aspect, the RNP complex can be pre-formed prior to injection into atarget cell or can be injected or introduced separately.

Also provided for, and included, in the present specification areisolated guide RNAs that can comprise a spacer selected from the groupconsisting of the first 20 nucleotides of each of SEQ ID NOs: 22 to 271and the first 20 nucleotides of each of SEQ ID NOs: 347 to 425. In anaspect, the Cas protein can be a protein comprising SEQ ID NOs: 20 or21.

The present specification also includes, and provides for, DNA vectorsthat can encode guide RNAs for the preparation of CRISPR/Cas RNPs. Ingeneral, a vector encoding a guide RNA can comprise a sequence selectedfrom the group consisting of the first 20 nucleotides of each of SEQ IDNOs: 22 to 271 or SEQ ID NOs: 347 to 425 and a guide RNA backbonearranged in cis and as part of a single transcription unit. Uponexpression in a suitable cell, the DNA vector can produce an sgRNA ofthe present specification. Suitable expression vector backbones,including promoters, selectable markers and replication origins, arewell known to persons of skill in the art. In practice, the 20nucleotides and the guide RNA backbone can be DNA (when expressed from apromoter to be transcribed in vivo (in cells) or in vitro (via T7polymerase) to form an RNA guide or the backbone can be chemicallysynthesized dual (crRNA and trRNA) guide or single guide RNA.

Specific guideRNAs can be paired to excise parts of the CD163 gene byusing paired guideRNAs to delete sections of the gene located betweenthose guides. Exemplary pairs of guideRNAs (listed as DNA sequences) areprovided in Table 5. Guide pairs without an exon 7 amino acid sequencelisted remove the entire exon. The complete amino acid sequence forthese deletions is set forth in SEQ ID NO: 553. All guides in Table 5can create the desired sequence by excising the DNA in between the twoguides and NHEJ repair between the cut sites without the use of a DNArepair template. The guide pairs that result in the amino acid sequencesset forth in SEQ ID NOs. 506-517 can introduce an exogenous stop codonacross the cut ends of the DNA introduced by the guide.

TABLE 5 Exemplary Guide RNA Pairs SEQ SEQ Exon ID ID Deletion Repaired7AA NO: Cut site NO: Cut site size Sequence SEQ (5′) (5′) (3′) (3′) (bp)SEQ ID ID 229 63322816 256 63323377 561 426 N/A 230 63322814 25663323377 562 427 N/A 231 63322826 256 63323377 551 428 N/A 237 63322861256 63323377 516 429 N/A 241 63322891 256 63323377 486 430 N/A 22963322816 258 63323378 562 431 N/A 230 63322814 258 63323378 563 432 N/A231 63322826 258 63323378 552 433 N/A 237 63322861 258 63323378 517 434N/A 241 63322891 258 63323378 487 435 N/A 229 63322816 261 63323373 558436 N/A 230 63322814 261 63323373 559 437 N/A 231 63322826 261 63323373548 438 N/A 237 63322861 261 63323373 513 439 N/A 241 63322891 26163323373 483 440 N/A 219 63322697 256 63323377 680 441 N/A 221 63322709256 63323377 668 442 N/A 224 63322747 256 63323377 630 443 N/A 22763322786 256 63323377 591 444 N/A 219 63322697 258 63323378 681 445 N/A221 63322709 258 63323378 669 446 N/A 224 63322747 258 63323378 631 447N/A 227 63322786 258 63323378 592 448 N/A 219 63322697 261 63323373 677449 N/A 221 63322709 261 63323373 665 450 N/A 224 63322747 261 63323373627 451 N/A 227 63322786 261 63323373 588 452 N/A 249 63322963 25663323377 414 453 N/A 250 63322973 256 63323377 404 454 N/A 249 63322963258 63323378 415 455 N/A 250 63322973 258 63323378 405 456 N/A 24963322963 261 63323373 411 457 N/A 250 63322973 261 63323373 401 458 N/A351 63323023 365 63323103 80 459 506 351 63323023 387 63323235 212 460506 348 63323027 390 63323236 209 461 507 348 63323027 388 63323236 209462 507 348 63323027 395 63323284 257 463 507 352 63323035 365 6332310368 464 508 352 63323035 387 63323235 200 465 508 352 63323035 39963323283 248 466 508 353 63323038 365 63323103 65 467 509 353 63323038387 63323235 197 468 509 353 63323038 399 63323283 245 469 509 35463323039 390 63323236 197 470 509 354 63323039 388 63323236 197 471 509354 63323039 395 63323284 245 472 509 358 63323056 361 63323077 21 473510 358 63323056 362 63323087 31 474 510 358 63323056 368 63323124 68475 510 358 63323056 384 63323203 147 476 510 358 63323056 394 63323272216 477 510 358 63323056 399 63323283 227 478 511 359 63323057 39063323236 179 479 511 359 63323057 388 63323236 179 480 511 359 63323057395 63323284 227 481 511 360 63323058 368 63323124 66 482 511 36063323058 384 63323203 145 483 511 360 63323058 389 63323237 179 484 511360 63323058 394 63323272 214 485 511 360 63323058 397 63323285 227 486511 361 63323077 365 63323103 26 487 512 361 63323077 387 63323235 158488 512 362 63323087 390 63323236 149 489 513 362 63323087 388 63323236149 490 512 362 63323087 395 63323284 197 491 513 364 63323089 36563323103 14 492 514 364 63323089 387 63323235 146 493 514 364 63323089399 63323283 194 494 514 365 63323103 368 63323124 21 495 515 36563323103 384 63323203 100 496 516 365 63323103 389 63323237 134 497 516365 63323103 394 63323272 169 498 516 365 63323103 397 63323285 182 499516 366 63323118 368 63323124 6 500 517 366 63323118 384 63323203 85 501517 366 63323118 389 63323237 119 502 517 366 63323118 394 63323272 154503 517 366 63323118 397 63323285 167 504 517 354 63323039 211 63323163123 505 518

In aspects, the guide RNA-Cas protein pairs can further comprise a DNArepair template. Exemplary guides and repair templates creating stopcodons are listed in Table 6. These and other guides can be paired withother repair templates to generate in-frame stop codons or perturb,interfere, or eliminate splicing of exons, as examples of disruptingCD163 mRNA translation or processing. Other examples can include, butare not limited to, the elimination of the start ATG codon, orprogramming repair outcomes when using paired guide RNAs where a repairtemplate could promote a single repair outcome over randomnon-homologous end joining when paired nucleases remove an exon (e.g.,deletion of exon 7 corresponding to domain 5) in CD163. Without beinglimited by theory, analysis reveals consistent reduction ofbioinformatic mismatches with inclusion of the extra nG in the PAM(nGGnG) as well as reduced off-target cutting in vitro and in vivo.Reduction of mismatches can include those from an alignment perspective(whether the DNA sequence has mismatches with the RNA guide sequence).See Table 4. A mismatch may not be an off-target edit (cutting orpresence of indel, in vitro or in vivo, respectively), but an off-targetedit is likely due to a mismatch. In the most preferred aspects, themethods disclosed herein produce no off-target edits in pigs, and thepigs disclosed herein have no off-target edits in their genomes.

TABLE 6 Guide RNAs and repair templates for editing Exon 2 of CD163Region Chr5 Location of 63301 63301 63303 63303 63303 63303 63303 6330363303 63306 63306 63306 63323 guideRNAs 998- 997- 213- 283- 315- 315-338- 379- 379- 148- 263- 364- 147- binding site 02017 02016 03232 0330203334 03334 03357 03398 03398 06167 06282 06383 23166 Repair tem-  1  2 3  4  5  6  7  8  9  10  11  12  13 plate SEQ ID NO Guide RNA 274 275276 277 278 279 280 281 282 283 284 285 286 spacer (DNA) SEQ ID NO GuideRNA 287 288 289 290 291 292 293 294 295 296 297 298 299 spacer (RNA) SEQID NO WT Region 300 301 302 303 304 305 306 307 308 309 310 311 312 SEQID NO. Trans-lation 313 314 315 316 317 318 319 320 321 322 323 324 325of WT region Translation of 326 327 328 329 330 331 332 333 334 335 336337 338 Deletion Base deletion chr5: chr5: chr5: chr5: chr5: chr5: chr5:chr5: chr5: chr5: chr5: chr5: chr5: coordinates 6330 6330 6330 6330 63306330 6330 6330 6330 6330 6330 6330 6332 1999- 1999- 3234- 3304- 3336-3337- 3351- 3399- 3400- 6156- 6282- 6373- 3159- 6330 6330 6330 6330 63306330 6330 6330 6330 6330 6330 6330 6332 2005 2005 3235 3307 3336 33373356 3401 3402 6158 6284 6377 3162 Deleted bases CTT CTT GG GGC G (1) G(1) CTT GGG GGG TTC CTC CGA CGG (#) GGT GGT (2) T (4) GTC (3) (3) (3)(3) TC C (4) C (7) C (7) (6) (5)

The stochastic natural cellular repair process of non-homologous endjoining (NHEJ) repair sometimes involves inserting or deleting single ormultiple nucleotides at the double-strand break. As a consequence ofthis repair process, if a double-strand break occurs in the codingregion of a gene, a shift in the translational reading frame of theencoded mRNA can result until an in-frame stop codon, terminatingprotein translation. Although the translation of this naturally,accidentally mutated gene can produce a shortened protein relative tothe protein product of an unmodified version, the amino acid sequence ofthis newly encoded polypeptide would be unique, possibly reducing oreven improving the fitness of cells within the target organism. In anaspect, to preclude the creation of frame shifting mutations and thetranslation of this set of undesirable polypeptides, edited pigs can bescreened for formation of an in-frame translational stop codon at ornear the endonuclease cut site that is a consequence of a separate,naturally occurring NHEJ repair.

The excision of genomic DNA sequence using two intronic guideRNAs inconjunction with an endonuclease can be accomplished by NHEJ repairsthat include, but are not limited to, the end-to-end joining of nucleasecut sites. Because introns are non-coding, NHEJ repair outcomes thatinclude indels around the nuclease cut sites can accomplish the excisionof the intended region of DNA which can include intron and/or exonsequences. Because some NHEJ repair outcomes occur more frequently thanothers, it is advantageous to consider repair outcome frequency whenchoosing a guideRNA pair for use in gene editing experiments. SEQ ID NO:520-555 illustrate repair outcomes of exon 7 excisions observed inblastocysts from guide pairs as set forth in Table 7. The designedrepair outcome for these guide pairs is listed in Table 5.

TABLE 7 Observed Repair Outcomes in Blastocysts SEQ ID NO: SEQ ID NO:Repair outcome (5′) (3′) SEQ ID 249 261 520 249 261 521 249 261 522 249261 523 249 261 524 249 261 525 249 261 526 249 261 527 249 256 528 249256 529 249 256 530 249 256 531 249 256 532 249 256 533 249 256 534 249256 535 241 258 536 241 258 537 241 258 538 241 258 539 241 258 540 241258 541 241 258 542 241 258 543 241 258 544 221 261 545 221 261 546 221261 547 221 261 548 221 261 549 229 256 550 229 256 551 229 256 552 229256 553 229 256 554 229 256 555

In another aspect, during gene editing, in-frame stop codons that do notresult in the addition of new amino acids can be created by including aDNA repair template together with the guide RNA-Cas protein pair. In anaspect, a DNA repair template can be a dsDNA. In another aspect, the DNArepair template can be a ssDNA. Co-introduction of a double- orsingle-stranded DNA repair template can be used to either delete orinsert DNA nucleotides to form an in-frame translational stop codon(TAA, TGA, TAG) at or near the double-strand break site initiated by theendonuclease. DNA repair templates can further comprise polynucleotidemodification templates containing several nucleotide changes incomparison to the native sequence, which can directly edit the targetDNA sequence and can be co-transfected with the endonuclease editingreagents to generate edited CD163 genes with in-frame stop codons. In anaspect, the encoded proteins from these gene edited CD163 genes, whentranscribed into mRNA and then translated into protein, can onlysynthesize a shortened and non-functional form of the CD163 polypeptide.Not to be limited by theory, DNA repair templates having longer regionsof sequence homology can be more efficient. In certain embodiments, DNArepair templates can contain regions of sequence identity (homologyarms) within the DNA repair template which can flank the sequence changeand can range from fewer than 50 nucleotides to greater than 1000nucleotides.

Several aspects of Cas9-protein/guide RNA combinations together with DNArepair templates are shown in Table 6. As provided in Table 6, severalCas9-protein/guide RNA combinations can be paired with DNA repairtemplates having, but not being limited to, 75 bases of sequencehomology on each side of the targeted deletion (50 core homology basesand flanking homology regions of 50 on either side). In an aspect, theprotein-guide RNA complexes and template combinations can be transfectedinto cells. Without being limited by theory, repair of the double-strandbreak using the DNA repair template can direct the formation of anin-frame translational stop codon as the result of deletion of single ormultiple nucleotides at or adjacent to the break site. Animals and cellsobtained from this method demonstrate that endonuclease-directeddouble-strand breaks at the porcine CD163 gene can be repaired using aco-introduced DNA repair template. As provided herein, repair templateshaving a sequence of SEQ ID NOs: 1 to 13 can introduce an in-frametranslational stop codon in Exon 2, thereby producing a shortened andnon-functional CD163 protein.

Use of a DNA repair template according to the present specification isnot limited to abolishing the function of CD163. The presentspecification further includes, and provides for, a repair template thatcan direct the removal or addition of nucleotides to the gene. In anaspect, the stability or half-life modulation of the encoded CD163 mRNAcan be modulated by editing according to the present methods. In yetanother aspect, DNA sequences which encode amino acids of the matureprotein responsible for binding PRRS virus can be removed or replaced.In an aspect, CD163 expression and or activity can be reduced by atleast 90% but not abolished. In an aspect of the present teachings,using the spacer sequences described with other repair templates wouldbe contemplated by those skilled in the art to allow for the formationof an in frame stop codon by the removal of bases and the introductionof bases or a combination thereof.

Included, and provided for, by the present specification are pigsderived from elite porcine lines comprising edited CD163 genes. Inaspects, the elite porcine lines can be PIC™ Line 15, PIC™ Line 17, PIC™Line 27, PIC™ Line 65, PIC™ Line 14, PIC™ Line 62, PIC337, PIC800,PIC280, PIC327, PIC408, PIC™ 399, PIC410, PIC415, PIC359, PIC380,PIC837, PIC260, PIC265, PIC210, PIC™ Line 2, PIC™ Line 3, PIC™ Line 4,PIC™ Line 5, PIC™ Line 18, PIC™ Line 19, PIC™ Line 92, PIC95, PIC™CAMBOROUGH® (Pig Improvement Company, Limited, Basingstoke, UK),PIC1070, PIC™ CAMBOROUGH® 40, PIC™ CAMBOROUGH® 22, PIC1050, PIC™CAMBOROUGH® 29, PIC™ CAMBOROUGH® 48, or PIC™ CAMBOROUGH® ×54. In variousaspects, the elite porcine lines can be PIC™ elite porcine lines 2, 3,15, 19, 27, 62, or 65. In another aspect, the pigs can comprise editedCD163 genes derived from elite porcine lines. In aspects, the pigs cancomprise an edited CD163 in a CD163 genomic region having the genotypesshown in Table 8 to Table 14. Table 8 to Table 14 present each positionon chromosome 5 in the vicinity of the CD163 gene in which a singlenucleotide polymorphism exists.

Each table presents alleles that are homozygous in that line andprovides a distinguishing genetic signature of the line, as well as itsunique genome edited region. All the genetic signatures are based on theSscrofa11.1 reference genome (GenBank accession: GCA_000003025.6).Accordingly, the transmission of the edited CD163 line can be followed,and animals comprising an edited CD163 gene obtained from the line canbe identified. Pigs of other lines are heterozygous or opposite genotypeat multiple alleles when compared to the lines of Table 8 to Table 14.In this way, the combination of genetic signatures in each line can beused to distinguish between pigs belonging to a particular line and pigsnot belonging to a particular line. In an aspect, the genetic signaturesprovide for methods to breed and track the CD163 edited genomes fromgeneration to generation. In an aspect, progeny generations thatcomprise the CD163 edited genomes having a genetic signature accordingto Table 8 to Table 14 can be prepared.

TABLE 8 Line 2 Genetic Signatures Position Genotype 60306428 C/C60306527 C/C 60308030 C/C 60320580 C/C 60322529 A/A 60324162 T/T60327987 C/C 60328009 T/T 60335421 T/T 60338654 G/G 60344946 A/A60345163 C/C 60345689 G/G 60345715 G/G 60345722 A/A 60345749 G/G60345775 A/A 60345825 A/A 60346409 G/G 60346607 A/A 60346641 G/G60346691 T/T 60346734 A/A 60347297 G/G 60350295 C/C 60350343 G/G60350448 C/C 60350470 T/T 60350475 C/C 60350564 A/A 60350571 A/A60350572 A/A 60350911 C/C 60351055 G/G 60351604 C/C 60351855 T/T60351857 C/C 60351972 G/G 60352165 C/C 60352923 C/C 60353408 A/A60353562 T/T 60353576 C/C 60353659 T/T 60353721 G/G 60354428 G/G60354925 C/C 60355415 G/G 60355420 T/T 60355448 G/G 60355529 A/A60355530 A/A 60355774 C/C 60356277 C/C 60356351 C/C 60356575 C/C60356578 T/T 60356861 G/G 60356885 T/T 60356898 T/T 60356914 C/C60357001 T/T 60357014 T/T 60358409 C/C 60358449 G/G 60358469 T/T60358475 T/T 60358551 A/A 60358568 G/G 60358641 T/T 60358704 T/T60358774 T/T 60368913 C/C 60368916 A/A 60368921 T/T 60368940 G/G60368963 C/C 60416097 G/G 60543479 C/C 60614797 G/G 60614964 G/G60920926 G/G 61479206 C/C 61479353 T/T 61653091 C/C 61783688 A/A62091204 A/A 62416522 A/A 62476273 C/C 62687883 C/C 63044336 A/A63546615 C/C 63553656 G/G 63558087 G/G 63640722 G/G 63792071 T/T64009226 C/C 64459105 T/T 64460101 A/A 64527707 A/A 64577968 C/C64943306 T/T 65242696 T/T 65242725 A/A 65242729 A/A 65242736 G/G65249484 T/T 65260283 T/T 65269510 C/C 65273886 T/T 65276133 C/C65277053 T/T 65277156 C/C 65277320 G/G 65280282 A/A 65282683 C/C65283434 C/C 65283970 C/C 65284829 C/C 65285100 C/C 65285902 G/G65285914 T/T 65286400 C/C 65294497 T/T 65383805 C/C 65405986 C/C65410118 C/C 65410147 G/G 65410198 C/C 65410435 C/C 65410440 T/T65410441 C/C 65410443 C/C 65410447 C/C 65411265 C/C 65411431 G/G65411524 C/C 65411698 G/G 65411831 C/C 65411848 G/G 65411854 T/T65411966 C/C 65412729 G/G 65413215 G/G 65413759 C/C 65417199 A/A65417256 G/G 65417261 A/A 65417273 C/C 65417287 G/G 65417716 G/G65417797 C/C 65417800 C/C 65419169 G/G 65419410 G/G 65420017 T/T65420184 G/G 65420415 A/A 65420591 G/G 65420680 C/C 65420681 A/A65420693 G/G 65420947 G/G 65420949 T/T 65421133 G/G 65421195 G/G65421255 G/G 65421365 G/G 65421421 C/C 65422005 G/G 65422008 G/G65422057 T/T 65422141 C/C 65422701 C/C 65422725 C/C 65425730 G/G65601310 C/C 65602328 C/C 65711602 C/C 65761990 C/C 66293766 G/G66296268 A/A

TABLE 9 Line 3 Genetic Signatures Position Genotype 60414864 T/T60415813 C/C 60415854 G/G 60572371 A/A 60663464 C/C 60663499 A/A61016527 G/G 61442674 T/T 61870484 C/C 61989476 G/G 62510031 G/G62602514 T/T 63044336 A/A 63345577 G/G 63452493 A/A 63669675 G/G63754659 T/T 63810627 C/C 63810632 G/G 63810707 T/T 63810733 G/G63810819 C/C 63810857 C/C 63810861 G/G 64010555 T/T 64010599 A/A64452338 T/T 64455982 C/C 64457532 T/T 64458163 T/T 64458711 G/G64555715 G/G 64561148 G/G 64561209 G/G 64561659 G/G 64561828 T/T64561873 G/G 64561935 C/C 64562021 C/C 64562771 C/C 64563462 G/G64682243 C/C 64682929 G/G 64685072 T/T 64692673 G/G 64692905 T/T64693058 T/T 64705228 G/G 64720067 A/A 64722426 T/T 64722539 C/C64726936 G/G 64729340 C/C 64730596 G/G 64736752 G/G 64761650 G/G64767369 G/G 64768411 G/G 64769978 T/T 64769989 C/C 64770307 G/G64773053 A/A 64774093 G/G 64775512 G/G 64775565 C/C 64780089 C/C64780152 C/C 64780196 C/C 64783169 G/G 64783774 C/C 64783896 G/G64784000 T/T 64784139 C/C 64784235 T/T 64784410 A/A 64784653 A/A64784725 C/C 64784844 A/A 64784875 T/T 64785001 A/A 64785078 C/C64813032 G/G 64819974 C/C 64893099 G/G 64899922 A/A 64934758 C/C65348500 G/G 65546268 A/A 65546327 G/G 65841270 T/T 65858531 G/G65979134 G/G 65985520 C/C 66035868 A/A 66042409 C/C 66042542 C/C66042551 C/C 66042557 A/A 66042563 G/G 66155827 A/A 66155859 A/A66155863 T/T 66156160 A/A 66156183 G/G 66156533 G/G 66167582 C/C66167809 C/C 66167823 T/T 66167877 A/A 66167888 C/C 66167905 A/A66168135 C/C 66168261 C/C 66168311 G/G 66168615 G/G 66168688 G/G66168742 G/G 66168880 G/G 66168947 T/T 66168952 A/A 66168993 A/A

TABLE 10 Line 15 Genetic Signatures Position Genotype 60391943 G/G60392234 T/T 60394009 A/A 60414796 T/T 60601475 A/A 60630898 A/A60630910 A/A 60786421 G/G 60787517 G/G 60792231 G/G 60795724 C/C60915104 C/C 61016227 A/A 61086145 T/T 61275101 C/C 61396087 A/A61434042 G/G 61515967 T/T 61759785 C/C 61759821 C/C 61865586 A/A61867498 C/C 62192420 G/G 62195570 A/A 62196101 C/C 62442547 G/G62509196 T/T 62675268 G/G 62843969 G/G 62852334 A/A 63025151 A/A63025152 C/C 63168616 A/A 63266682 T/T 63367266 G/G 63424311 T/T63535058 T/T 63653754 G/G 63655538 T/T 63669242 A/A 63669944 C/C63669946 G/G 63900332 A/A 64138082 C/C 64171653 C/C 64471845 G/G64472363 A/A 64472504 T/T 64472616 T/T 64523343 A/A 64536599 G/G64537982 T/T 64893205 A/A 64916422 G/G 65039734 C/C 65175496 C/C65177506 G/G 65294016 C/C 65571397 A/A 65573608 T/T 65576707 C/C65984917 G/G 65984984 T/T 65985058 T/T 65985392 C/C 66042472 C/C66099282 A/A 66119075 T/T

TABLE 11 Line 19 Genetic Signatures Position Genotype 60320100 A/A60320144 G/G 60320580 C/C 60507836 C/C 60512716 G/G 60564418 A/A60666662 T/T 60683738 T/T 60782197 T/T 60782278 T/T 60782642 A/A60782683 G/G 60782756 G/G 60782997 G/G 60783031 C/C 60783198 G/G61131519 G/G 61131736 C/C 61189029 C/C 61359517 A/A 61390748 C/C61465215 T/T 61567329 C/C 61567357 A/A 61567365 A/A 61567410 C/C61567494 C/C 61567594 G/G 61975156 G/G 61975367 G/G 61975382 A/A61975388 C/C 62363513 T/T 62367647 G/G 62378299 G/G 62610431 T/T62952267 T/T 62965473 T/T 63108711 G/G 63109224 T/T 63174233 T/T63174257 A/A 63393845 G/G 63393849 T/T 63393851 T/T 63860532 A/A63861247 A/A 63862368 T/T 64138082 C/C 64171653 C/C 64181448 G/G64402245 T/T 64455172 G/G 64514012 C/C 64514013 A/A 64522169 G/G64522173 T/T 64522177 T/T 64522178 C/C 64794635 G/G 65031621 C/C65031630 C/C 65031684 T/T 65222383 T/T 65222385 A/A 65242693 C/C65497229 G/G 65499233 C/C 65502398 G/G 65562917 C/C 65566225 T/T65567066 T/T 65894449 C/C 65904537 T/T 65959573 A/A 65998062 C/C66158553 A/A 66158950 C/C 66159103 A/A 66159212 G/G

TABLE 12 Line 27 Genetic Signatures Position Genotype 60347934 A/A60356984 C/C 60692943 C/C 60693260 G/G 60693769 G/G 60938885 T/T60960728 A/A 61436306 G/G 61464114 C/C 61468375 G/G 61736415 A/A61736429 G/G 61757961 G/G 61820110 A/A 61822513 T/T 61858837 G/G62159353 T/T 62313847 C/C 62315464 A/A 62570257 C/C 62570793 A/A62571313 T/T 62917536 G/G 62917597 T/T 62917618 C/C 62917629 T/T62917780 G/G 62917866 T/T 62917889 T/T 62918448 G/G 62918456 A/A62918465 A/A 62918655 T/T 63042154 C/C 63043261 G/G 63046692 G/G63433705 G/G 63490990 A/A 63626018 A/A 63626423 T/T 63626826 C/C63630149 A/A 63631098 C/C 63977777 A/A 63977821 G/G 64460204 C/C64582243 C/C 64582246 G/G 64582272 A/A 64600735 G/G 65056396 A/A65056671 T/T 65332987 A/A 65335837 C/C 65390844 A/A 65404042 A/A65404045 A/A 65404063 C/C 65705261 G/G 65740018 T/T 65740030 A/A66235840 G/G

TABLE 13 Line 62 Genetic Signatures Position Genotype 60336383 A/A60343172 A/A 60345254 G/G 60447765 G/G 60452143 C/C 60452167 T/T60462769 T/T 60653700 T/T 60654939 A/A 60654943 A/A 61085770 A/A61108831 G/G 61117357 A/A 61234908 C/C 61331270 A/A 61388824 G/G61755575 C/C 61759095 C/C 61760230 C/C 61927492 T/T 61927506 A/A62006220 T/T 62115641 T/T 62169134 T/T 62368918 C/C 62471593 C/C62474001 A/A 62474006 C/C 62474365 A/A 62480042 A/A 62480261 G/G62480699 A/A 62480892 A/A 62481902 G/G 62482914 T/T 62483893 C/C62483897 T/T 62847072 G/G 62874766 G/G 62897778 A/A 63100290 C/C63219344 G/G 63361759 G/G 63388357 C/C 63442314 G/G 63590333 T/T63670724 G/G 63714834 A/A 63792005 T/T 63985848 A/A 63985923 G/G64131088 C/C 64471400 C/C 64483848 G/G 64484177 T/T 64689271 A/A64758235 C/C 64790188 C/C 64895383 C/C 64991645 A/A 65045805 T/T65146186 C/C 65368620 C/C 65432698 C/C 65542716 A/A 65637507 G/G65771614 C/C 65771615 G/G 65772162 T/T 66033248 T/T 66033476 C/C66033484 C/C

TABLE 14 Line 65 Genetic Signatures Position Genotype 60322300 C/C60411747 A/A 60412758 G/G 60412775 A/A 60412826 C/C 60413294 G/G60459719 A/A 60462179 C/C 60492928 A/A 60492938 T/T 60666662 T/T60722708 C/C 60770183 G/G 60770197 C/C 60777431 C/C 60781592 T/T60782197 T/T 60782278 T/T 60782642 A/A 60782683 G/G 60782756 G/G60782997 G/G 60783006 G/G 60783031 C/C 60783198 G/G 60791719 T/T60900261 G/G 60911551 A/A 60912669 A/A 61223574 A/A 61486607 C/C61756324 G/G 61832527 C/C 61895566 C/C 61895833 A/A 62354225 C/C62364072 G/G 62383525 G/G 62399452 T/T 62404734 A/A 62432982 C/C62841388 G/G 62931496 T/T 63156406 C/C 63156417 T/T 63266198 A/A63383925 A/A 63392495 T/T 63549670 C/C 63593491 G/G 63609938 A/A63641998 C/C 63678793 G/G 63784770 G/G 63810897 C/C 63810903 C/C63810920 G/G 63810922 T/T 63810928 G/G 63872757 C/C 63944636 G/G63944765 C/C 64138639 C/C 64194054 G/G 64242768 T/T 64270989 G/G64753488 G/G 64758888 A/A 64758900 C/C 64759492 T/T 64795760 T/T64797970 A/A 64798690 G/G 64860395 G/G 64860396 C/C 64862131 C/C65371202 T/T 65371472 G/G 65371901 G/G 65371970 C/C 65372072 A/A65409493 A/A 65437347 G/G 65573362 T/T 65601655 G/G 65747069 T/T65843843 A/A 65849424 T/T 66248756 A/A

Elite PIC™ lines 2, 3, 15, 19, 27, 62 and 65 are lines selected forsuperior commercial phenotypes. In an aspect, the CD163 gene editedcells and animals can be free of deleterious alleles that are present inwild populations and in many commercial herds. In aspects, CD163 geneedited cells and animals can be free of one of more of the deleteriousalleles selected from the group consisting of epetheliogenesisimperfecta, melanotic skin tumors, dermatosis vegetans, abnormal mamae,shortened vertebral column, kinky tail, rudimentary tail, Hairlessness,Hairlessness (2), Woolly hair, Hydrocephalus, Tassels, Legless,Three-legged, Syndactyly, Polydactyly, Pulawska factor, Heterochromiairidis, Congenital tremor A III, Congenital tremor A IV, Congenitalataxia, Hind leg paralysis, Bentleg, Thickleg, Malignant hyperthermia,Hemophilia (von Willebrand's disease), Leukemia, Hemolytic disease,edema, Acute respiratory distress (“barker”), Rickets, 25 Renalhypoplasia, Renal cysts, Uterus aplasia, Porcine Stress Syndrome (PSS),halothane (HAL), Dipped Shoulder (Humpy Back, Kinky Back, Kyphosis),Hyperostosis, Mammary Hypoplasia, and Undeveloped Udder. As providedherein, the improved methods of preparing CD163 gene edited animals canavoid introducing new mutations at the deleterious loci or generatingnew amorphic, hypomorphic, hypermorphic, neomorphic, antimorphicmutations at non-target sites. These latter changes can be particularlyundesirable in elite lines as they can interfere with genes involved indesirable traits that can be controlled by multiple loci in acontinuous, quantitative way in a population. Many such QuantitativeTrait Loci (QTL) are known and can be typically characterized by abell-shaped curve the trait value can be plotted against the number ofobserved animals. Such polygenic inheritance of traits can be commonamong traits recognized as commercially important such as, but notlimited to, backfat, average daily feed intake, lifetime daily gain, andloin depth.

Similarly, traits associated with productivity can be multigenic andcontrolled by multiple QTLs. These traits were typically measured byvisual inspection but methods now can include ultrasound to measurebackfat thickness (bfp), loin depth (ldp) and intramuscular fat (uip).As provided in Table 15, elite lines can have desirable phenotypictraits including high backfat and loin depth while having high lifetimedaily gain. Similarly, the sows of the elite lines can be fecund andhave large litters, few still-born, and sufficient teats to ween andnurse the piglets.

TABLE 15 Desirable Phenotypic Traits standard LINE TRT average deviation2 Backfat, mm 7.8 1.81 3 Backfat, mm 9.44 2.77 15 Backfat, mm 8.16 2.0365 Backfat, mm 7.45 1.88 2 Average Daily Feed Intake, kg 1.99 0.24 3Average Daily Feed Intake, kg 2.13 0.25 15 Average Daily Feed Intake, kg2.22 0.24 65 Average Daily Feed Intake, kg 2.2 0.27 2 Lifetime DailyGain, grams/day 683.83 73.53 3 Lifetime Daily Gain, grams/day 704.2380.15 15 Lifetime Daily Gain, grams/day 755.61 69.32 65 Lifetime DailyGain, grams/day 800.6 84.48 2 Loin depth, mm 65.31 7.09 3 Loin depth, mm62.61 7.14 15 Loin depth, mm 67.17 7.15 65 Loin depth, mm 81.2 8 2 TotalBorn per litter 13.71 3.19 3 Total Born per litter 15.06 3.41 15 TotalBorn per litter 10.39 2.71 65 Total Born per litter 10.31 2.52 2 Stillborn per litter 1.19 1.43 3 Still born per litter 1.25 1.50 15 Stillborn per litter 1.22 1.37 65 Still born per litter 0.77 1.16 2 Teatnumber 15.43 1.27 3 Teat number 15.44 1.29

The present specification provides for, and includes, gene edited pigsof selected elite lines that can be homozygous for CD163 knockout edits(CD163^(−/−)). In an aspect, the line can be selected from the groupconsisting of PIC™ Line 2, Line 3, Line 15, Line 19, Line 27, Line 62,Line 65, and progeny thereof comprising the edited CD163 genes describedherein. Gene edited lines 2, 3, 15, 19, 27, 62, and 65 can compriseCD163 genomic regions as provided at Table 8 to Table 14, and can bereadily distinguished from each other, from unimproved lines, and fromother elite lines. In aspects according to the present specification,the heterozygous and homozygous pigs of the present specification can befree of off-site mutations. The present specification provides for, andincludes, pigs and cells that can have edited CD163 genes comprising SEQID NOs: 1 to 18 and 426 to 505 and that can share a genetic signaturecomprising at least 90% of the genotypic markers of Table 8 to Table 14.The present specification provides for, and includes, pigs and cellsthat can have edited CD163 genes comprising SEQ ID NO: 2 and sharing agenetic signature comprising at least 90% of the genotypic markers ofTable 8 to Table 14. In an aspect, the genetic signature of a CD163edited pig or cell can share a genetic signature comprising at least 95%of the genotypic markers of Table 8 to Table 14. Also included are CD163edited pigs or cells that can share a genetic signature comprising atleast 97% of the genotypic markers of Table 8 to Table 14. In an aspect,the CD163 edited pigs or cells can share a genetic signature comprisingat least 98% of the genotypic markers of Table 8 to Table 14. Anotheraspect provides for CD163 edited pigs or cells that can share a geneticsignature comprising at least 99% of the genotypic markers of Table 8 toTable 14.

As provided herein, the gene-edited CD163^(−/−) animals and cells ofLines 2, 3, 15, 19, 27, 62, and 65, can retain desirable commercialtraits and can be free of deleterious off-target mutations and cancomprise an edited CD163 gene comprising any of SEQ ID NOs: 1 to 18 and426-505. In aspects of the present disclosure, the gene-editedCD163^(−/−) animals and cells of Lines 2, 3, 15, 19, 27, 62, and 65, cancomprise at least 90% of the loin depth of the non-edited pig line andcan comprise an edited CD163 gene comprising any of SEQ ID NOs: 1 to 18and 426-505. In aspects of the present disclosure, the gene-editedCD163^(−/−) animals and cells of Lines 2, 3, 15, 19, 27, 62, and 65, cancomprise at least 90% of the lifetime daily gain of the non-edited pigline. In aspects of the present disclosure, the gene-edited CD163^(−/−)animals and cells of Lines 2, 3, 15, 19, 27, 62, and 65, can comprise atleast 90% of the average daily feed intake of the non-edited pig line.The present specification provides for, and includes, pigs and cellsthat can have edited CD163 genes comprising SEQ ID NOs: 1 to 18 and 426to 505 and can share a genetic signature comprising at least 90% of thegenotypic markers of Table 8 to Table 14.

As provided herein, the gene-edited CD163^(−/−) animals and cells ofLines 2, 3, 15, 19, 27, 62, and 65, can retain desirable reproductivetraits. In aspects of the present disclosure, the gene-editedCD163^(−/−) animals and cells of Lines 2, 3, 15, 19, 27, 62, and 65,comprising a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505can comprise at least 90% of the total born per litter of the non-editedpig line. In aspects of the present disclosure, the gene-editedCD163^(−/−) animals and cells of Lines 2, 3, 15, 19, 27, 62, and 65,comprising a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505can comprise no more than 110% of the number of still born pigletscompared to the non-edited pig line. In aspects of the presentdisclosure, the gene-edited CD163^(−/−) animals and cells of Lines 2, 3,15, 19, 27, 62, and 65, comprising a gene-edited CD163^(−/−) of SEQ IDNOs:1 to 18 or 426 to 505 can comprise at least 90% of the averagenumber of teats compared to the non-edited pig line.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 2having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line 2animals. In an aspect, gene edited CD163^(−/−) animals and cells of Line2 can have backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 3having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line 3animals. In an aspect, gene edited CD163^(−/−) animals and cells of Line3 can have backfat that is at least 95% of the unedited Line 3 animal.In an aspect, gene edited CD163^(−/−) animals and cells of Line 3 canhave backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 15having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line15 animals. In an aspect, gene edited CD163^(−/−) animals and cells ofLine 15 can have backfat that is at least 95% of the unedited Line 15animal. In an aspect, gene edited CD163^(−/−) animals and cells of Line15 can have backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 19having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line19 animals. In an aspect, gene edited CD163^(−/−) animals and cells ofLine 19 can have backfat that is at least 95% of the unedited Line 19animal. In an aspect, gene edited CD163^(−/−) animals and cells of Line19 have can backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 27having a gene-edited CD163^(−/−) of SEQ ID NOs: 1 to 18 or 426 to 505can have backfat that is at least 90% of the amount found in uneditedLine 27 animals. In an aspect, gene edited CD163^(−/−) animals and cellsof Line 27 can have backfat that is at least 95% of the unedited Line 27animal. In an aspect, gene edited CD163^(−/−) animals and cells of Line27 can have backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 62having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line62 animals. In an aspect, gene edited CD163^(−/−) animals and cells ofLine 62 can have backfat that is at least 95% of the unedited Line 62animal. In an aspect, the gene edited CD163^(−/−) animals and cells ofLine 62 having a gene-edited CD163^(−/−) of SEQ ID NO: 2 can havebackfat that is at least 95% of the amount found in unedited Line 62animals. In an aspect, gene edited CD163^(−/−) animals and cells of Line62 can have backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 65having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave backfat that is at least 90% of the amount found in unedited Line65 animals. In an aspect, gene edited CD163^(−/−) animals and cells ofLine 65 can have backfat that is at least 95% of the unedited Line 65animal. In an aspect, gene edited CD163^(−/−) animals and cells of Line65 can have backfat that is at least 97% of the unedited Line 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 2having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave a number of total born per litter that is at least 90% of theamount found in unedited Line 2 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 2 can have a number of total bornper litter that is at least 95% of the unedited Line 2 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 2 can have anumber of total born per litter that is at least 97% of the uneditedLine 2 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 3having a gene-edited CD163^(−/−) of SEQ ID NOs: 1 to 18 or 426 to 505can have a number of total born per litter that is at least 90% of theamount found in unedited Line 3 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 3 can have a number of total bornper litter that is at least 95% of the unedited Line 3 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 3 can have anumber of total born per litter that is at least 97% of the uneditedLine 3 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 15having a gene-edited CD163^(−/−) of SEQ ID NOs: 1 to 18 or 426 to 505can have a number of total born per litter that is at least 90% of theamount found in unedited Line 15 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 15 can have a number of total bornper litter that is at least 95% of the unedited Line 15 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 15 can have anumber of total born per litter that is at least 97% of the uneditedLine 15 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 19having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave a number of total born per litter that is at least 90% of theamount found in unedited Line 19 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 19 can have a number of total bornper litter that is at least 95% of the unedited Line 19 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 19 can have anumber of total born per litter that is at least 97% of the uneditedLine 19 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 27having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave a number of total born per litter that is at least 90% of theamount found in unedited Line 27 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 27 can have a number of total bornper litter that is at least 95% of the unedited Line 27 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 27 can have anumber of total born per litter that is at least 97% of the uneditedLine 27 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 62having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave a number of total born per litter that is at least 90% of theamount found in unedited Line 62 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 62 can have a number of total bornper litter that is at least 95% of the unedited Line 62 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 62 can have anumber of total born per litter that is at least 97% of the uneditedLine 62 animal.

In an aspect, the gene edited CD163^(−/−) animals and cells of Line 65having a gene-edited CD163^(−/−) of SEQ ID NOs:1 to 18 or 426 to 505 canhave a number of total born per litter that is at least 90% of theamount found in unedited Line 65 animals. In an aspect, gene editedCD163^(−/−) animals and cells of Line 65 can have a number of total bornper litter that is at least 95% of the unedited Line 65 animal. In anaspect, gene edited CD163^(−/−) animals and cells of Line 65 can have anumber of total born per litter that is at least 97% of the uneditedLine 65 animal.

The present specification provides for and includes CD163 edited animalscomprising the germplasm of PIC™ Line 15, PIC™ Line 17, PIC™ Line 27,PIC™ Line 65, PIC™ Line 14, PIC™ Line 62, PIC337, PIC800, PIC280,PIC327, PIC408, PIC™ 399, PIC410, PIC415, PIC359, PIC380, PIC837,PIC260, PIC265, PIC210, PIC™ Line 2, PIC™ Line 3, PIC™ Line 4, PIC™ Line5, PIC™ Line 18, PIC™ Line 19, PIC™ Line 92, PIC95, PIC™ CAMBOROUGH®,PIC1070, PIC™ CAMBOROUGH® 40, PIC™ CAMBOROUGH® 22, PIC1050, PIC™CAMBOROUGH® 29, PIC™ CAMBOROUGH® 48, or PIC™ CAMBOROUGH® ×54. As usedherein, the term germplasm includes an intact genome present in cells ornuclei and comprising chromosomes. The term germplasm may include anygamete, germ cell, or any somatic cell from which an animal can becloned. The edited germplasm can comprise an edit having an editedgenomic sequence of any one of SEQ ID NOs: 426 to 505. The editedgermplasm can comprise an edit having an edited genomic sequence of anyone of SEQ ID NOs: 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505.The edited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 15. The edited germplasm can comprisea genome that is 80%, 85%, 90%, 95% similar or identical to PIC™ Line17. The edited germplasm can comprise a genome that is 80%, 85%, 90%,95% similar or identical to PIC™ Line 27. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 65. The edited germplasm can comprise a genome that is 80%,85%, 90%, 95% similar or identical to PIC™ Line 14. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 62. The edited germplasm can comprise a genome that is 80%,85%, 90%, 95% similar or identical to PIC337. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC800. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC280. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC327. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC408. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ 399. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC410. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC415. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC359. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC380. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC837. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC260. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC265. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC210. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC™ Line 2. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 3. The edited germplasm can comprise a genome that is 80%,85%, 90%, 95% similar or identical to PIC™ Line 4. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 5. The edited germplasm can comprise a genome that is 80%,85%, 90%, 95% similar or identical to PIC™ Line 18. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 19. The edited germplasm can comprise a genome that is 80%,85%, 90%, 95% similar or identical to PIC™ Line 92. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC95. The edited germplasm can comprise a genome that is 80%, 85%, 90%,95% similar or identical to PIC™ CAMBOROUGH®. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC1070. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC™ CAMBOROUGH® 40. The editedgermplasm can comprise a genome that is 80%, 85%, 90%, 95% similar oridentical to PIC™ CAMBOROUGH® 22. The edited germplasm can comprise agenome that is 80%, 85%, 90%, 95% similar or identical to PIC1050. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ CAMBOROUGH® 29. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ CAMBOROUGH® 48. The edited germplasm can comprise a genome that is80%, 85%, 90%, 95% similar or identical to PIC™ CAMBOROUGH® ×54.

The present specification provides for and includes CD163 edited animalscomprising the germplasm of PIC™ Line 2, Line 3, Line 15, Line 19, Line27, Line 62, or Line 65. The edited germplasm can comprise an edithaving an edited genomic sequence of any one of SEQ ID NOs: 426 to 505.The edited germplasm can comprise an edit having an edited genomicsequence of any one of SEQ ID NOs: 426, 427, 428, 429, 430, 431, 432,433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,503, 504, or 505. The edited germplasm can comprise an edit having anedited genomic sequence as set forth in SEQ ID NO: 453. The editedgermplasm can comprise a genome that is 80%, 85%, 90%, 95% similar oridentical to PIC™ Line 2 with a CD163 edited sequence comprising SEQ IDNO: 453. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to PIC™ Line 3 with a CD163 editedsequence comprising SEQ ID NO: 453. The edited germplasm can comprise agenome that is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 15with a CD163 edited sequence comprising SEQ ID NO: 453. The editedgermplasm can comprise a genome that is 80%, 85%, 90%, 95% similar oridentical to PIC™ Line 19 with a CD163 edited sequence comprising SEQ IDNO: 453. The edited germplasm can comprise a genome that is 80%, 85%,90%, 95% similar or identical to 80%, 85%, 90%, 95% similar or identicalto PIC™ Line 27 with a CD163 edited sequence comprising SEQ ID NO: 453.The edited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 62 with a CD163 edited sequencecomprising SEQ ID NO: 453. The edited germplasm can comprise a genomethat is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 65 with aCD163 edited sequence comprising SEQ ID NO: 453.

The edited germplasm can comprise an edit having an edited genomicsequence as set forth in SEQ ID NO: 489. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 2 with a CD163 edited sequence comprising SEQ ID NO: 489. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 3 with a CD163 edited sequencecomprising SEQ ID NO: 489. The edited germplasm can comprise a genomethat is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 15 with aCD163 edited sequence comprising SEQ ID NO: 489. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 19 with a CD163 edited sequence comprising SEQ ID NO: 489. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to 80%, 85%, 90%, 95% similar or identical to PIC™Line 27 with a CD163 edited sequence comprising SEQ ID NO: 489. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 62 with a CD163 edited sequencecomprising SEQ ID NO: 489. The edited germplasm can comprise a genomethat is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 65 with aCD163 edited sequence comprising SEQ ID NO: 489. The edited germplasmcan have a predicted exon 7 amino acid sequence from any one of SEQ IDNOs: 506-517. The edited germplasm can have a predicted exon 7 aminoacid sequence as set forth in SEQ ID NO: 513.

The edited germplasm can comprise an edit having an edited genomicsequence as set forth in SEQ ID NO: 505. The edited germplasm cancomprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 2 with a CD163 edited sequence comprising SEQ ID NO: 505. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 3 with a CD163 edited sequencecomprising SEQ ID NO: 505. The edited germplasm can comprise a genomethat is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 15 with aCD163 edited sequence comprising SEQ ID NO: 505. The edited germplasmcan comprise a genome that is 80%, 85%, 90%, 95% similar or identical toPIC™ Line 19 with a CD163 edited sequence comprising SEQ ID NO: 505. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to 80%, 85%, 90%, 95% similar or identical to PIC™Line 27 with a CD163 edited sequence comprising SEQ ID NO: 505. Theedited germplasm can comprise a genome that is 80%, 85%, 90%, 95%similar or identical to PIC™ Line 62 with a CD163 edited sequencecomprising SEQ ID NO: 505. The edited germplasm can comprise a genomethat is 80%, 85%, 90%, 95% similar or identical to PIC™ Line 65 with aCD163 edited sequence comprising SEQ ID NO: 505. The edited germplasmcan have a predicted exon 7 amino acid sequence as set forth in SEQ IDNO: 518.

The present specification also provides for and includes cells of PIC™Line 2, Line 3, Line 15, Line 19, Line 27, Line 62, or Line 65. In someembodiments, a cell of PIC™ Line 2 can comprise an edited genomicsequence of any one of SEQ ID NOs: 426 to 505. The cell of PIC™ Line 2can comprise an editing genomic sequence as set forth in SEQ ID NO: 453.In some embodiments, a cell of PIC™ Line 3 can comprise an editedgenomic sequence of any one of SEQ ID NOs: 426 to 505. The cell of PIC™Line 3 can comprise an editing genomic sequence as set forth in SEQ IDNO: 453. In some embodiments, a cell of PIC™ Line 15 can comprise anedited genomic sequence of any one of SEQ ID NOs: 426 to 505. The cellof PIC™ Line 15 can comprise an edited genomic sequence as set forth inSEQ Id NO: 453. In some embodiments, a cell of PIC™ Line 19 can comprisean edited genomic sequence of any one of SEQ ID NOs: 426 to 505. Thecell of PIC™ Line 19 can comprise an editing genomic sequence as setforth in SEQ Id NO: 453. In some embodiments, a cell of PIC™ Line 27 cancomprise an edited genomic sequence of any one of SEQ ID NOs: 426 to505. The cell of PIC™ Line 27 can comprise an editing genomic sequenceas set forth in SEQ ID NO: 453. In some embodiments, a cell of PIC™ Line62 can comprise an edited genomic sequence of any one of SEQ ID NOs: 426to 505. The cell of PIC™ Line 62 can comprise an editing genomicsequence as set forth in SEQ Id NO: 453. In some embodiments, a cell ofPIC™ Line 65 can comprise an edited genomic sequence of any one of SEQID NOs: 426 to 505. The cell of PIC™ Line 65 can comprise an editinggenomic sequence as set forth in SEQ Id NO: 453. The edited germplasmcan have a predicted exon 7 amino acid sequence as set forth in SEQ IDNO: 513.

The present specification provides for, and includes, hybrid animalsthat can comprise the CD163 gene edits characterized by SEQ ID NOs: 1 to18 and 426 to 505. In some configurations, gene edit can comprise SEQ IDNO: 453. In an aspect, the hybrid animal can be a CD163^(−/−) hybridanimal of the CAMBOROUGH® (PIC™ UK Limited, Basingstoke, UK) line.CAMBOROUGH® pigs are hybrids that can be prepared by a cross betweenLine 2 and Line 3. In an aspect, the hybrid animal can be a CD163^(−/−)hybrid animal of the CAMBOROUGH® line having an edited CD163 gene of SEQID NO: 2. In an aspect, the hybrid animal can be a CD163^(−/−) hybridanimal of the CAMBOROUGH® line having an edited CD163 gene of SEQ ID NO:426-458. In various aspects, the hybrid animal can be a CD163^(−/−)hybrid animal of the CAMBOROUGH® line having an edited CD163 gene of SEQID NO: 453. In an aspect, the hybrid animal can be a CD163^(−/−) hybridanimal of the CAMBOROUGH® line having an edited CD163 gene of SEQ ID NO:459-504. In various aspects, the hybrid animal can be a CD163^(−/−)hybrid animal of the CAMBOROUGH® line having an edited CD163 genesequence of SEQ ID NO: 489. CAMBOROUGH® hybrid pigs are pigs that havelarge litters with uniform and vigorous piglets. CAMBOROUGH® hybrid pigshave a long productive life and have a low mortality. CAMBOROUGH®CD163^(−/−) hybrid pigs retain these desirable commercial traits. In anaspect, CAMBOROUGH® CD163^(−/−) hybrid pigs have litter sizes that areindistinguishable from non-gene edited CAMBOROUGH® hybrid pigs. Inaspects according to the present specification, the heterozygous andhomozygous pigs of the present specification are free of off-sitemutations.

The present specification further provides, and includes, methods forpreparing CD163 gene edited hybrid animals. In an aspect, a first parentcan comprise a CD163 gene edited boar, gilt, or sow of one of PIC™ Line2, Line 3, Line 15, Line 19, Line 27, Line 62, or Line 65 for crossingto a second parent. In an aspect, the genome of a gene edited firstparent can comprise a sequence selected from the group consisting of SEQID NOs: 1 to 18 or 426 to 505. In some aspects, the genome can compriseSEQ ID NO: 453. In various aspects, the genome can comprise SEQ ID NO:489. In various aspects, the genome can comprise SEQ ID NO: 505. In anaspect, the method can comprise a second parent selected from the groupconsisting of a CD163 gene edited boar, gilt, or sow of one of PIC™ Line2, Line 3, Line 15, Line 19, Line 27, Line 62, or Line 65. Also providedfor, and included, by the present specification are methods of preparingCD163 edited animals that can comprise crossing a progeny of any one ofPIC™ Line 2, Line 3, Line 15, Line 19, Line 27, Line 62, or Line 65.

In an aspect, the present specification provides for heterozygous pigsfrom lines 2, 3, 15, and 65 (Table 8, Table 9, Table 10, and Table 14,respectively) that can have at least one copy of the CD163 genesuccessfully edited in a knock-out edit. These edited pigs can exhibit ahealthy phenotype with no noticeable deleterious effects from the edit.In another aspect, the specification provides for pigs from lines 19,27, and 62 (Table 11, Table 12, and Table 13, respectively) that can beedited using the methods disclosed herein to generate edited pigs withhealthy phenotypes. The heterozygous pigs can be crossed with non-editedanimals of the corresponding line to produce F1 heterozygous pigs. In anembodiment, heterozygous pigs of lines 2, 3, 15, and 65 can be crossedto a second heterozygous pig of lines 2, 3, 15, and 65, and homozygousCD163 edited pigs can be produced in Mendelian proportions. Notably, thegene edits can be unique and identifiable using SEQ ID NOs: 1 to 18 and426 to 505 and share genetic signatures comprising at least 90% of thegenotypic markers of Table 8 to Table 14, thereby enabling the detectionand breeding of the CD163 gene edited genomes in any progeny generation.In an aspect, the genetic signatures can share 95% or more of thegenotypic markers of Table 8 to Table 14. In another aspect, the geneticsignatures share 97% or more of the genotypic markers of Table 8 toTable 14. Also included are genetic signatures that can share 98% ormore of the genotypic markers of Table 8 to Table 14. Importantly, thelinkage of the CD163 edits to the genomic regions identifiable using thegenotypic markers of Table 8 to Table 14 can enable the preparation ofany progeny animal having the desired edits and the tracking of theedited regions in any number of progeny generations. In aspectsaccording to the present specification, the heterozygous and homozygouspigs of the present specification can be free of off-site mutations.

The present specification provides for, and includes, an embryo orzygote that can be obtained from an elite line of pigs. In an aspect,the embryo or zygote can be obtained from an elite porcine line selectedfrom the group consisting of PIC™ Line 2, Line 3, Line 15, Line 19, Line27, Line 62, or Line 65. In an aspect, the embryo or zygote can be afrozen embryo or zygote. In another aspect, the embryo or zygote can bea frozen blastocyst. As provided herein, the embryos can be preparedfrom in vitro matured oocytes collected from estrus synchronized gilts.The surrounding cumulus cells can be removed from the in vitro maturedoocytes and incubated with washed boar spermatozoa and incubated. Afterincubation, presumptive zygotes can be microinjected with an RNP mixturecomprising the CRISPR-Cas endonuclease and guide RNA combinationscomprising the first 20 nucleotides of SEQ ID NOs: 22 to 271 or 347 to425 listed in Table 3. Injected embryos can be transferred to asurrogate female at the 1 to 4 cell stage. In an aspect, the RNP mixturecan further include repair templates listed in Table 6 (SEQ ID NOs: 1 to13). In an aspect, injected zygotes can be surgically implanted into theoviducts of estrus synchronized, un-mated surrogate females by amid-line laparotomy under general anesthesia (each surrogate receives40-60 injected embryos).

The present specification provides for, and includes, gene edited pigsof selected elite lines that can be CD163^(−/−). In an aspect, the linecan be PIC™ Line 15, PIC™ Line 17, PIC™ Line 27, PIC™ Line 65, PIC™ Line14, PIC™ Line 62, PIC337, PIC800, PIC280, PIC327, PIC408, PIC™ 399,PIC410, PIC415, PIC359, PIC380, PIC837, PIC260, PIC265, PIC210, PIC™Line 2, PIC™ Line 3, PIC™ Line 4, PIC™ Line 5, PIC™ Line 18, PIC™ Line19, PIC™ Line 92, PIC95, PIC™ CAMBOROUGH®, PIC1070, PIC™ CAMBOROUGH® 40,PIC™ CAMBOROUGH® 22, PIC1050, PIC™ CAMBOROUGH® 29, PIC™ CAMBOROUGH® 48,or PIC™ CAMBOROUGH® ×54. In an aspect, the line can be selected from thegroup consisting of PIC™ Line 2, Line 3, Line 15, Line 19, Line 27, Line62, Line 65, and progeny thereof comprising the edited CD163 genesdescribed herein. Gene edited lines 2, 3, 15, 19, 27, 62, and 65comprise CD163 genomic regions as provided above at Table 8 to Table 14,and can be readily distinguished from each other, from unimproved lines,and from other elite lines. Similarly, progeny of lines 2, 3, 15, 19,27, 62, and 65 comprising the CD163^(−/−) genomic regions as provided inTable 8 to Table 14 can be identified. Accordingly, the presentspecification provides for progeny pigs that can have a CD163^(−/−)genomic region.

The present specification provides for and includes hybrid porcine linescomprising an edited CD163 gene of the present teachings. In someaspects, the hybrid porcine line can be produced by crossing an editedPIC™ line with at least one other edited PIC™ line. In some aspects, theporcine line can be produced by serial crosses to introduce germplasmfrom three or more porcine lines. In an aspect, the line can be PIC™Line 15, PIC™ Line 17, PIC™ Line 27, PIC™ Line 65, PIC™ Line 14, PIC™Line 62, PIC337, PIC800, PIC280, PIC327, PIC408, PIC™ 399, PIC410,PIC415, PIC359, PIC380, PIC837, PIC260, PIC265, PIC210, PIC™ Line 2,PIC™ Line 3, PIC™ Line 4, PIC™ Line 5, PIC™ Line 18, PIC™ Line 19, PIC™Line 92, PIC95, PIC™ CAMBOROUGH®, PIC1070, PIC™ CAMBOROUGH® 40, PIC™CAMBOROUGH® 22, PIC1050, PIC™ CAMBOROUGH® 29, PIC™ CAMBOROUGH® 48, orPIC™ CAMBOROUGH® ×54.

In various aspects, PIC™ Line 65 is sold under the trade name PIC337. Invarious aspects, PIC™ line 62 is sold under the tradename PIC408. Invarious aspects, hybrid pigs made by crossing PIC™ lines 15 and 17 aresold under the tradenames PIC800 or PIC280. In various aspects, PIC™Line 27 is sold under the tradename PIC327. In various aspects, hybridscreated from crossing PIC™ Line 65 and PIC™ Line 62 is sold under thetradenames PIC399, PIC410 or PIC415. In various aspects, hybrids createdfrom crossing PIC™ Line 65 and Pic Line 27 are sold under the tradenamePIC359. In various aspects, hybrids prepared from crossing PIC™ Line 800pigs (which is a hybrid of PIC™ Line 15 and PIC™ Line 17) to PIC™ Line65 pigs are sold under the tradenames PIC380 or PIC837. In variousaspects, PIC™ Line 14 is sold under the trade name PIC260. In variousaspects, hybrids created from crossing PIC™ Line 14 and PIC™ Line 65 aresold under the tradename PIC265. In various aspects, hybrids created bycrossing PIC™ Line 2 and PIC™ Line 3 are sold under the tradenamesPIC210, PIC™ CAMBOROUGH®, and PIC1050. In various aspects, hybrids ofPIC™ Line 3 and PIC™ Line 92 are sold under the tradename PIC95. Invarious configurations, hybrids made from crossing PIC™ Line 19 and PIC™Line 3 are sold under the tradename PIC1070. In various aspects, hybridscreated by crossing PIC™ Line 18 and PIC™ Line 3 are sold under thetradename PIC™ CAMBOROUGH® 40. In various aspects, hybrids created fromcrossing PIC™ Line 19 and PIC1050 (which is itself a hybrid of PIC™lines 2 and 3) are sold under the tradename PIC™ CAMBOROUGH® 22. Invarious aspects, hybrids created from crossing PIC™ Line 2 and PIC1070(which is itself a hybrid of PIC™ lines 19 and 3) are sold under thetradename PIC™ CAMBOROUGH® 29. In various aspects, hybrids created fromcrossing PIC™ Line 18 and PIC1050 (which is itself a hybrid of PIC™lines 2 and 3) are sold under the tradename PIC™ CAMBOROUGH® 48. Invarious aspects, hybrids created from crossing PIC™ Line 4 and PIC™ Line5 are sold under the tradename PIC™ CAMBOROUGH® ×54. The presentteachings provide for and include pigs of any of the forgoing lines orhybrids comprising a CD163 edit of the present teachings.

In various aspects, the present teachings provide for an include pigscomprising an edited CD163 gene comprising an edited sequence set forthin SEQ ID NOs: 453, 489, or 505 wherein the pig can be a pig of PIC™Line 15, PIC™ Line 17, PIC™ Line 27, PIC™ Line 65, PIC™ Line 14, PIC™Line 62, PIC™ Line 2, PIC™ Line 3, PIC™ Line 4, PIC™ Line 5, PIC™ Line18, PIC™ Line 19, PIC™ Line 92, or a hybrid of two or more of theselines. In various aspects, the hybrid pig can be a cross of PIC™ Line 15and PIC™ Line 17, PIC™ Line 65 and PIC™ Line 62, PIC™ Line 65 and PIC™Line 27, a serial hybrid of PIC™ Line 15 and PIC™ Line 17, wherein thehybrid offspring is then crossed to PIC™ Line 65, PIC™ Line 14 and PIC™Line 65, PIC™ Line 2 and PIC™ Line 3, PIC™ Line 3 and PIC™ Line 92, PIC™Line 19 and PIC™ Line 3, PIC™ Line 18 and PIC™ Line 3, a serial hybridbetween a hybrid pig of PIC™ Line 2 and PIC™ Line 3 and a pig of PIC™Line 19, a serial hybrid between a hybrid pig of PIC™ Line 19 and PIC™Line 3 and a pig of PIC™ Line 2, a serial hybrid between a hybrid ofPIC™ Line 2 and PIC™ Line 3 crossed to a pig of PIC™ Line 18, or ahybrid of PIC™ Line 4 and PIC™ Line 5.

In various aspects, the hybrid line comprising an edited CD163 gene canbe produced by crossing two or more of lines 2, 3, 15, 19, 27, 62, or65. In some aspects, the hybrid line comprising an edited CD163 gene canbe a CAMBOROUGH® line. CAMBOROUGH® pigs are hybrids that can be preparedby a cross between Line 2 and Line 3. In various aspects, the hybridline can comprise a PIC™ 837 hybrid line comprising an edited CD163gene. PIC™ 837 pigs are hybrids that can be prepared by crossing PIC™Line 800 pigs to PIC™ Line 65 pigs.

The present specification provides for, and includes, hybrid CD163^(−/−)progeny lines comprising one CD163^(−/−) genomic region that can beobtained from lines 2, 3, 15, 19, 27, 62, or 65, and a secondCD163^(−/−) genomic region that can be obtained from a different lineselected from lines 2, 3, 15, 19, 27, 62, or 65. In an aspect, a hybridCD163^(−/−) progeny line can comprise a CD163^(−/−) allele in a genomicregion according to Table 8 and a CD163^(−/−) allele in a genomic regionaccording to Table 10. In an aspect, a hybrid CD163^(−/−) progeny linecan comprise a CD163^(−/−) allele in a genomic region according to Table8 and a CD163^(−/−) allele in a genomic region according to Table 14. Inan aspect, a hybrid CD163^(−/−) progeny line can comprise a CD163^(−/−)allele in a genomic region according to Table 9 and a CD163^(−/−) allelein a genomic region according to Table 10. In an aspect, a hybridCD163^(−/−) progeny line can comprise a CD163^(−/−) allele in a genomicregion according to Table 9 and a CD163^(−/−) allele in a genomic regionaccording to Table 14.

The present specification provides for, and includes, hybrid animalsthat can comprise the CD163 gene edits characterized by SEQ ID NOs: 1 to18 and 426 to 505. In an aspect, the hybrid animals comprising the CD163gene edits can be characterized by SEQ ID NO: 2. In an aspect, thehybrid animals comprising the CD163 gene edits can be characterized bySEQ ID NO: 426-458. In an aspect, the hybrid animals comprising theCD163 gene edits can be characterized by SEQ ID NO: 453. In an aspect,the hybrid animals comprising the CD163 gene edits can be characterizedby SEQ ID NO: 459-504. In an aspect, the hybrid animals comprising theCD163 gene edits can be characterized by SEQ ID NO: 489. In an aspect,the hybrid animal can be a CD163^(−/−) hybrid animals of the CAMBOROUGH®line. CAMBOROUGH® pigs are hybrids that can be prepared by a crossbetween Line 2 and Line 3.

In various aspects, the hybrid pig can comprise SEQ ID NO: 2 in agenomic region according to Table 8 and SEQ ID NO: 2 in a genomic regionaccording to Table 10. In an aspect, the hybrid pig can comprise SEQ IDNO: 453 in a genomic region according to Table 8 and SEQ ID NO: 453 in agenomic region according to Table 10. In an aspect, the hybrid pig cancomprise SEQ ID NO: 489 in a genomic region according to Table 8 and SEQID NO: 489 in a genomic region according to Table 10. In an aspect, ahybrid CD163^(−/−) progeny line can comprise SEQ ID NO: 2 in a genomicregion according to Table 8 and SEQ ID NO: 2 in a genomic regionaccording to Table 14. In an aspect, a hybrid CD163^(−/−) progeny linecan comprise SEQ ID NO: 453 in a genomic region according to Table 8 andSEQ ID NO: 453 in a genomic region according to Table 14. In an aspect,a hybrid CD163^(−/−) progeny line can comprise SEQ ID NO: 489 in agenomic region according to Table 8 and SEQ ID NO: 489 in a genomicregion according to Table 14. In an aspect, a hybrid CD163^(−/−) progenyline can comprise SEQ ID NO: 2 in a genomic region according to Table 9and SEQ ID NO: 2 in a genomic region according to Table 10. In anaspect, a hybrid CD163^(−/−) progeny line can comprise SEQ ID NO: 453 ina genomic region according to Table 9 and SEQ ID NO: 453 in a genomicregion according to Table 10. In an aspect, a hybrid CD163^(−/−) progenyline can comprise SEQ ID NO: 489 in a genomic region according to Table9 and SEQ ID NO: 489 in a genomic region according to Table 10. In anaspect, the hybrid pig can comprise SEQ ID NO: 2 in a genomic regionaccording to Table 8 and SEQ ID NO: 2 in a genomic region according toTable 10. In an aspect, the hybrid pig can comprise SEQ ID NO: 453 in agenomic region according to Table 8 and SEQ ID NO: 453 in a genomicregion according to Table 10. In an aspect, the hybrid pig can compriseSEQ ID NO: 489 in a genomic region according to Table 8 and SEQ ID NO:489 in a genomic region according to Table 10. In an aspect, a hybridCD163^(−/−) progeny line can comprise SEQ ID NO: 2 in a genomic regionaccording to Table 8 and SEQ ID NO: 2 in a genomic region according toTable 14. In an aspect, a hybrid CD163^(−/−) progeny line can compriseSEQ ID NO: 453 in a genomic region according to Table 8 and SEQ ID NO:453 in a genomic region according to Table 14. In an aspect, a hybridCD163^(−/−) progeny line can comprise SEQ ID NO: 489 in a genomic regionaccording to Table 8 and SEQ ID NO: 489 in a genomic region according toTable 14. In an aspect, a hybrid CD163^(−/−) progeny line can compriseSEQ ID NO: 2 in a genomic region according to Table 9 and SEQ ID NO: 2in a genomic region according to Table 10. In an aspect, a hybridCD163^(−/−) progeny line can comprise SEQ ID NO: 453 in a genomic regionaccording to Table 9 and SEQ ID NO: 453 in a genomic region according toTable 10. In an aspect, a hybrid CD163^(−/−) progeny line can compriseSEQ ID NO: 489 in a genomic region according to Table 9 and SEQ ID NO:489 in a genomic region according to Table 10. In aspects according tothe present specification, the heterozygous and homozygous pigs of thepresent specification can be free of off-site mutations.

In an aspect, the hybrid animal can be a CD163^(−/−) hybrid animals ofthe CAMBOROUGH® line. CAMBOROUGH® pigs are hybrids that can be preparedby a cross between Line 2 and Line 3. In an aspect, the hybrid pig cancomprise SEQ ID NO: 426 to 505 in a genomic region according to Table 8and SEQ ID NO: 426-505 in a genomic region according to Table 10. In anaspect, the hybrid pig can comprise SEQ ID NO: 453 in a genomic regionaccording to Table 8 and SEQ ID NO: 453 in a genomic region according toTable 10. In an aspect, the hybrid pig can comprise SEQ ID NO: 489 in agenomic region according to Table 8 and SEQ ID NO: 489 in a genomicregion according to Table 10. In an aspect, a hybrid CD163^(−/−) progenyline can comprise SEQ ID NO: 426 to 505 in a genomic region according toTable 8 and SEQ ID NO: 426 to 505 in a genomic region according to Table14. In an aspect, a hybrid CD163^(−/−) progeny line can comprise SEQ IDNO: 453 in a genomic region according to Table 8 and SEQ ID NO: 489 in agenomic region according to Table 14. In an aspect, a hybrid CD163^(−/−)progeny line can comprise SEQ ID NO: 489 in a genomic region accordingto Table 8 and SEQ ID NO: 453 in a genomic region according to Table 14.In an aspect, a hybrid CD163^(−/−) progeny line can comprise SEQ ID NO:426 to 505 in a genomic region according to Table 9 and SEQ ID NO: 426to 505 in a genomic region according to Table 10. In an aspect, a hybridCD163^(−/−) progeny line can comprise SEQ ID NO: 453 in a genomic regionaccording to Table 9 and SEQ ID NO: 453 in a genomic region according toTable 10. In an aspect, a hybrid CD163^(−/−) progeny line can compriseSEQ ID NO: 489 in a genomic region according to Table 9 and SEQ ID NO:489 in a genomic region according to Table 10.

In various aspects, an edited pig of PIC™ line 2, PIC™ line 3, PIC™ line15, PIC™ line 19, PIC™ line 27, PIC™ line 62, or PIC™ line 65 can have aCD163 gene comprising a predicted amino acid sequence as set forth inSEQ ID NO: 518. In various aspects, the edited CD163 gene can have a 123bp deletion as set forth in SEQ ID NO: 505. In various configurations,this deletion can have an Exon 7 Amino Acid sequence of AHRKPRLV - - -TVVSLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVV CS (SEQ ID NO:518). In various aspects, a pig having an edited CD163 gene comprisingan amino acid sequence set forth in SEQ ID NO: 518 can be a hybridoffspring between two PIC™ lines. In various aspects, the pig can havean edited CD163 Exon 7 sequence comprising a nucleotide sequence setforth in SEQ ID NO: 505. In various aspects, the pig can be an offspringof a cross between PIC™ line 2 and PIC™ line 3. In aspects according tothe present specification, the heterozygous and homozygous pigs of thepresent specification can be free of off-site mutations.

Importantly, the CD163 edited pigs and cells of the presentspecification retain their desirable commercial phenotypes. The editedpigs can exhibit a healthy phenotype with no noticeable acutedeleterious effects from the edit. In an aspect, the pigs of lines 2, 3,15, 19, 27, 62, and 65, can retain the commercially desirable phenotypesas provided in Table 15.

Methods for improving the health of existing herds of livestock cancomprise modifying the CD163 gene locus using the methods describedabove. In an aspect, the method can comprise introducing into a pig cellan endonuclease or a polynucleotide encoding said endonuclease, and aguide polynucleotide comprising a sequence selected from the groupconsisting of the first 20 nucleotides of each of SEQ ID NOs: 22 to 271or the first 20 nucleotides of each of 347 to 425, incubating the cellunder conditions that permit the endonuclease to act upon the DNA at, ornear, the target sequence and thereby induce recombination,homology-directed repair, or non-homologous end joining at or near thetarget site, identifying at least one cell having a modification at saidtarget sequence, and producing an animal from an animal cell. In aspectsof the present specification, the endonuclease can be an RNP complex ofa guide RNA and a Cas protein from S. thermophilus. In another aspect,the endonuclease can be an RNP complex of a guide RNA and a Cas proteinfrom S. pyogenes.

In an aspect, the method can further comprise providing to the cell arepair guide comprising a sequence selected from the group consisting ofnucleotides 50 to 100 of SEQ ID NOs: 1 to 13. In another aspect, arepair guide can comprise a sequence selected from the group consistingof nucleotides 50 to 100 of SEQ ID NOs: 1 to 13 and can further comprise85% homology to nucleotides 1 to 49 and 101 to 150 of SEQ ID NOs: 1 to13. In an aspect, the repair guide can comprise a sequence selected fromthe group consisting of SEQ ID NOs: 1 to 13. As provided herein, themethod for improving the health of existing herds provides for themaintenance of desirable commercial phenotypes as discussed above. In anaspect, the desirable commercial phenotypes can be at least 90% of thephenotypes observed in herds of non-edited pigs having a similar geneticbackground.

The CD163 gene edited locus can be introduced into the herd byconventional breeding or by methods incorporating artificialinsemination. To prepare homozygous animals, crosses between parentshaving at least one gene edited CD163 locus comprising a sequence of SEQID NOs: 1 to 18 or 426 to 505 can be performed and homozygous progeny(present at a 1:4 Mendelian ratio) can be selected. As provided herein,a combination of parents and one gene edited CD163 locus can be suitablefor improving the herd. Further breeding of animals having the CD163gene edited loci can improve the health of the herd until all theanimals of the herd comprise a CD163 gene edited locus as described.Notably, the health of the herd can improve significantly well beforethe herd has been bred fully to a CD163 gene edited herd. Specifically,and as discussed above, pig fetuses in a PRRSv resistant gene editedCD163 sow are themselves protected from PRRSv. Further, one of ordinaryskill in the art would know that, as the numbers of homozygous CD163edited animals increase, suitable pig vectors for PRRSv transmissiondecrease (e.g., herd immunity develops). Accordingly, the presentmethods can provide for improving a herd by introducing herd immunity.

One useful method of detecting the desired edit is to use real-time PCR.PCR primers flanking the region of interest and a probe thatspecifically anneals to the region of interest. The probe is labelledwith both a fluorophore and a quencher. In the PCR reaction, the primersand probe hybridize in a sequence-dependent manner to the complementaryDNA strand of the region of interest. Because the probe is intact, thefluorophore and quencher are in close proximity and the quencher absorbsfluorescence emitted by the fluorophore. The polymerase extends from theprimers and begins DNA synthesis. When the polymerase reaches the probe,the exonuclease activity of the polymerase cleaves the hybridized probe.As a result of cleavage, the fluorophore is separated from the quencherand fluoresces. This fluorescence is detected by the real timeinstrument. These steps are repeated for each PCR cycle and allowdetection of specific products.

In the instant application, three separate sets of primers and probeswere designed. The first set of primers (SEQ ID NO: 556 and 557) flankedthe unedited genomic sequence comprising SEQ ID NO: 249 and a probe (SEQID NO: 558) which binds to the unedited genomic DNA in between theprimers. The second set of primers (SEQ ID NO: 559 and 560) flanked theunedited genomic sequence comprising SEQ ID NO: 256, and a probe (SEQ IDNO: 561) binds the unedited between the primers. The final set ofprimers (SEQ ID NO: 562 and 563) flanked the desired Exon 7 deletionedit created by excision of the sequence between the cut sites of SEQ IDNO: 249 and SEQ ID NO: 256. A probe (SEQ ID NO: 564) was designed tobind the desired edit in between these primers. A commercial real-timePCR kit was then used to probe various animals for the desired edit. Avariety of commercial real-time PCR kits exist including, such as, butwithout limitation, PRIMETIME® from IDT, TAQMAN® (Roche MolecularSystems, Inc, Pleasonton, Calif.) from Applied Biosystems, and variouskits from Qiagen and Bio-Rad. Skilled persons will recognize that anysuch kit can be used with the primers and methods of the presentteachings to achieve like results.

EXAMPLES Example 1

This Example illustrates target site selection of a porcine CD163 geneknockout in pig cells.

A Streptococcus Cas9/gRNA RNA-directed DNA endonuclease was used togenerate DNA sequence insertions, deletions, and combinations thereof,in the porcine CD163 gene (Sscrofa11.1, GenBank accession: GCA000003025.6), whereby guide RNAs and protein combinations were deliveredsingly or as pairs, such that sequence changes in the CD163 gene reduceor abolish the function, stability, or expression of the CD163 messengerRNA or protein. The sequence of the CD163 gene from 120 nucleotidesupstream (chr5:63300192) of the translational start site to 59nucleotides downstream of CD163 exon 7 (chr5:63323390), was screened forthe guide RNA binding sites having either an adjacent nGG or nGGnG PAMsequence required for cutting by the Cas9 proteins derived fromStreptococcus pyogenes or Streptococcus thermophilus CRISPR3 (S.thermophilus CR3), respectively, the sequences of which are in thesequence listing filed herewith, as listed in Table 2. The DNA sequencesfor the target sites for editing, the locations of the target sites onthe CD163 gene, and the editing activity (measured as described inExample 3) are provided in Table 3. Guide RNA molecules had the samesequence as the target, with corresponding RNA nucleotides, without thePAM sequence (nGG for S. pyogenes and nGGnG for S. thermophilus). Guidepolynucleotide molecules could also consist of DNA bases or mixtures ofDNA and RNA bases. The target site sequences are listed in the sequencelisting filed herewith as SEQ ID NOs: 22 to 271 and 347 to 425. Thetarget site sequences shown are conserved across pig germplasm andscreened by DNA sequencing.

Example 2

This example illustrates nucleofection for the delivery of guideRNA/Cas9 endonuclease to porcine fetal fibroblasts.

To test the DNA cutting activity in living cells to produce an editedporcine CD163 allele, the CRISPR-Cas endonuclease and guide RNAtargeting the sequences listed in Table 3 were nucleofected into porcinefetal fibroblast cells. Porcine fetal fibroblast (PFF) cells lines wereprepared from 28-35 day-old fetuses and 3.2 μg of Cas9 protein (S.pyogenes or S. thermophilus) and 2.2 μg of in vitro transcribed singleguide RNA were combined in water to a total volume of 2.23 μl, then werenucleofected into PFF cells using a Lonza electroporator. In preparationfor nucleofection, PFF cells were harvested using TrypLE express(recombinant Trypsin), upon which the culture medium was removed fromcells, washed 1× with HBSS or DPBS, and incubated for 3-5 minutes at38.5° C. in the presence of TrypLE. Cells were then harvested withcomplete medium. Cells were pelleted via centrifugation (300×g for 5minutes at room temperature), supernatant was discarded, then the cellswere resuspended in 10 mL PBS to obtain single cell suspension countingcells using trypan blue staining.

The appropriate amount of cells was pelleted by centrifugation (300×gfor 5 minutes at room temperature), the supernatant was discarded, andthe cells were resuspended in nucleofection buffer P3 at a finalconcentration of 7.5×10⁶ cells/ml. 20 μl of the cell suspension wereadded to each well of a nucleofection plate containing the RNP mixtureusing a multichannel pipette, then mixed gently to resuspend the cells.The RNP/cell mixture was transferred in the nucleofection plate,nucleofected with program CM138 (supplied by the manufacturer). 80 μl ofwarm Embryonic Fibroblast Medium, EFM, (Dulbecco's Modified Eagle'sMedium (DMEM) containing 2.77 mM glucose, 1.99 mM L-glutamine, and 0.5mM sodium pyruvate, supplemented with 100 μM 2-Mercaptoethanol, 1×Eagle's minimum essential medium non-essential amino acids (MEM NEAA),100 μg/mL Penicillin-Streptomycin, and 12% Fetal Bovine Serum were addedto each well after nucleofection. The suspensions were mixed gently bypipetting, then 100 μl were transferred to a 12 well plate containing900 μl of EFM pre-incubated at 38.5° C. The plate was then incubated at38.5° C., 5% CO₂ for 48 hours. Forty-eight hours post nucleofection,genomic DNA was prepared from transfected and control PFF cells, 15 μlof QUICKEXTRACT™ DNA Extraction Solution (Lucigen, Madison, Wis.) wereadded to pelleted cells then lysed by incubating for 10 mins at 37° C.,for 8 mins at 65° C., for 5 mins at 95° C., then lysate was held at 4°C. until used for DNA sequencing.

Example 3

This example illustrates the editing frequency of guide RNA/Cas9combinations directed against porcine CD163.

Nucleotide sequence changes were introduced into the porcine CD163 geneby delivering Cas9 protein complexed with guide RNAs to fetal fibroblastcells as described in Example 2.

To evaluate DNA double strand cleavage at a porcine CD163 genomic targetsite mediated by the guide RNA/Cas endonuclease system, a region ofapproximately 250 bp genomic DNA surrounding the target site wasamplified by PCR and the PCR product was then examined by amplicon deepsequencing for the presence of edits. After transfection in triplicate,PFF genomic DNA was extracted as described in Example 2. The regionsurrounding the intended target site was PCR amplified with NEB Q5Polymerase, adding sequences necessary for amplicon-specific barcodesand ILLUMINA® (ILLUMINA®, San Diego, Calif.) sequencing using tailedprimers through two rounds of PCR. The resulting PCR amplifications weredeep sequenced on an ILLUMINA® MISEQ® Personal Sequencer (ILLUMINA®, SanDiego, Calif.). The resulting reads were examined for the presence ofedits at the expected site of cleavage by comparison to controlexperiments where the Cas9 protein and guide RNA were omitted from thetransfection or by comparison to the reference genome. To calculate thefrequency of NHEJ edits for a target site, Cas9 protein, guide RNAcombination, the total number of edited reads (amplicon sequencescontaining insertions or deletions when compared to the DNA sequencesfrom control treatments or reference genome) were divided by total readnumber (wild-type plus edited reads) of an appropriate length containinga perfect match to the barcode and forward primer. Total read countsaveraged approximately 7000 per sample and NHEJ activity is expressed asthe average (n=3) edited fraction in Table 3. As shown in Table 3, from0 to 58.2% of the reads contained edits and the average editingfrequency across all combinations was approximately 16%. This exampledemonstrates that, in fetal fibroblast cells, the porcine CD163 genenucleotide sequence was edited through the stimulation of double-strandbreaks mediated by transfecting Cas9 protein from either S. pyogenes orS. thermophilus complexed with various guide RNAs.

Example 4

This example illustrates generation of an in-frame stop codon using DNArepair template.

DNA repair templates were used to introduce stop codons in the porcineCD163 gene when co-delivered with Cas9 protein and guide RNA. Theendonuclease-guide complexes and repair templates disclosed in Table 5were generated and used to edit CD163 genes in blastocysts. The basedeletions were verified by the methods of Example 5.

Example 5

This example illustrates the molecular characterization of edited animalgenomes.

A tissue sample was taken from an animal whose genome was been editedaccording to the examples herein. Tail, ear notch, or blood samples weresuitable tissue types. The tissue sample was frozen at −20° C. within 1hour of sampling to preserve integrity of the DNA in the tissue sample.

DNA was extracted from tissue samples after proteinase K digestion inlysis buffer. Characterization was performed on two different sequenceplatforms, short sequence reads using the ILLUMINA® platform and longsequence reads on an Oxford NANOPORE™ platform (Oxford NANOPORE™Technologies, Oxford, UK).

For short sequence reads, two-step PCR was used to amplify and sequencethe region of interest. The first step was a locus-specific PCR whichamplified the locus of interest from the DNA sample using a combinedlocus-specific primer with a vendor-specific primer. The second stepattached the sequencing index and adaptor sequences to the amplicon fromthe first step so that sequencing could occur.

The locus-specific primers for the first step PCR were chosen so thatthey amplified a region <300 bp such that ILLUMINA® paired-endsequencing reads could span the amplified fragment. Multiple ampliconswere preferred to provide redundancy should deletions or naturallyoccurring point mutations prevent primers from correctly binding.Sequence data for the amplicon was generated using an ILLUMINA®sequencing platform (MISEQ®, ILLUMINA®, San Diego, Calif.). Sequencereads are analyzed to characterize the outcome of the editing process.

For long sequence reads, two-step PCR was used to amplify and sequencethe region of interest. The first step is a locus-specific PCR whichamplified the locus of interest from the DNA sample using a combinedlocus-specific primer with a vendor-specific adapter. The second stepPCR attached the sequencing index to the amplicon from the first-stepPCR so that the DNA was ready for preparing a sequencing library. Thestep 2 PCR products underwent a set of chemical reactions from a vendorkit to polish the ends of the DNA and ligate on the adapter containingthe motor protein to allow access to the pores for DNA strand-basedsequencing.

The locus specific primers for the first step PCR range were designed toamplify different regions of the CD163 gene and amplified regionsdiffered in length. Normalized DNA is then mixed with vendor suppliedloading buffer and is loaded onto the NANOPORE™ flowcell.

Long sequence reads, while having lower per base accuracy than shortreads, are very useful for observing the long range context of thesequence around the target site.

Example 6

This example illustrates methods of making pigs having edited CD163genes conferring PRRSv resistance.

Porcine oocytes were isolated, fertilized, and then the resultingzygotes are edited to generate gene edited pigs.

CD163 RNP complexes were microinjected into the cytoplasm of in vivo orin vitro fertilized porcine one-cell zygotes. These zygotes were thenincubated to generate edited multicellular embryos and transferred tosurrogate gilts via standard methods to birth gene edited pigs. Toprepare embryo donors and surrogates, pubertal gilts from PIC™ Line 2,Line 3, Line 15, and Line 65 were subjected to estrus synchronization bytreatment with 0.22% altrenogest solution (20-36 mg/animal) for 14 days.Follicular growth was induced by the administration of PMSG 36 hoursfollowing the last dose of Matrix, and ovulation was induced by theadministration of hCG 82 hours after PMSG administration. To generate invivo fertilized zygotes, females in standing heat were then artificiallyinseminated (AI) with boar semen from the corresponding PIC™ line. Invivo derived zygotes were recovered surgically 12-24 hours after AI byretrograde flushing the oviduct with sterile TL-HEPES mediumsupplemented with 0.3% BSA (w/v). Fertilized zygotes were subjected to asingle 2-50 picoliter (pl) cytoplasmic injection of Cas9 protein andguide RNA complex (25-50 ng/μl and 12.5-35 ng/μl) targeting CD163 andcultured in PZM5 medium (Yoshioka, K., et al., Biol. Reprod., 2002, 60:112-119; Suzuki, C., et al., Reprod. Fertil. Dev., 2006 18, 789-795;Yoshioka, K., J. Reprod. Dev. 2008, 54, 208-213). Injected zygotes weresurgically implanted into the oviducts of estrus synchronized, un-matedsurrogate females by a mid-line laparotomy under general anesthesia(each surrogate received 20-60 injected embryos).

In vitro fertilized embryos for gene editing were derived fromnon-fertilized PIC™ oocytes. Immature oocytes from estrus synchronizedPIC™ gilts were collected from medium size (3-6 mm) follicles. Oocyteswith evenly dark cytoplasm and intact surrounding cumulus cells werethen selected for maturation. Cumulus oocyte complexes were placed in awell containing 500 μl of maturation medium, TCM-199 (Invitrogen) with3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/mlEGF, 0.5 μg/ml luteinizing hormone (LH), 0.5 μg/ml FSH, 10 ng/mlgentamicin (Sigma), and 10% follicular fluid for 42-44 h at 38.5° C. and5% CO₂, in humidified air. At the end of the maturation, the surroundingcumulus cells were removed from the oocytes by vortexing for 3 min inthe presence of 0.1% hyaluronidase. Then, in vitro matured oocytes wereplaced in 100 μl droplets of IVF medium (modified Tris-buffered mediumcontaining 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl₂), 11 mM glucose, 20 mMTris, 2 mM caffeine, 5 mM sodium pyruvate, and 2 mg/ml bovine serumalbumin (BSA)) in groups of 25-30 oocytes and were fertilized accordingto established protocol (Abeydeera, Biol. Reprod., 57:729-734, 1997)using fresh extended boar semen. One ml of extended semen was mixed withDulbecco's Phosphate Buffered Saline (DPBS) containing 1 mg/ml BSA to afinal volume of 10 ml and centrifuged at 1000×g, 25° C. for 4 minutes,and spermatozoa were washed in DPBS three times. After the final wash,spermatozoa were re-suspended in mTBM medium and added to oocytes at afinal concentration of 1×10⁵ spermatozoa/ml, and co-incubated for 4-5 hat 38.5° C. and 5% CO₂. Presumptive zygotes were microinjected 5 hourspost IVF and transferred to a surrogate female after 18-42 hours (1-4cell stage). Each surrogate receives 20-60 injected embryos. Pregnancieswere confirmed by lack of return to estrus (21 days) and ultrasound at28 days post embryo transfer.

To establish the frequency of Cas9-guide RNA targeted gene editing inporcine embryos, uninjected control zygotes and injected surplus zygotesgenerated by in vitro fertilization were cultivated in PZM3 or PZM5medium at 38.5° C. for 5-7 days. Blastocysts were harvested at day 7post cultivation and the genomic DNA isolated for next generationsequencing.

Example 7

This example illustrates the generation and characterization of geneedited pigs.

Animals from PIC™ lines 2, 3, 15, and 65 were edited by the methodsdescribed in Example 6. Successful edits were confirmed using themethods of Example 5. Fibroblast cell lines were grown from collagenasetreated ear notch samples extracted from the edited animals anddeposited with the American Type Culture Collection (ATCC®). The ATCC®has an address of 10801 University Boulevard, Manassas, Va. 20110-2209.A representative sample of CD163 edited PIC™ Line 2 was deposited withthe ATCC on Apr. 3, 2019 and assigned ATTC® Patent Deposit NumberPTA-125814. A representative sample of CD163 edited PIC™ Line 3 wasdeposited with the ATCC on Apr. 3, 2019 and assigned ATTC® PatentDeposit Number PTA-125815. A representative sample of CD163 edited PIC™Line 15 was deposited with the ATCC on Apr. 3, 2019 and assigned ATTC®Patent Deposit Number PTA-125816. A representative sample of CD163edited PIC™ Line 65 was deposited with the ATCC® on Apr. 3, 2019 andassigned ATTC® Patent Deposit Number PTA-125813. Each deposit was madeaccording to the Budapest Treaty. Representative animals from each linewere confirmed to have heterozygous edits as specified in Table 16.

TABLE 16 Verified Gene Edited Animals Line number CD163 Allele 1 CD163Allele 2 ATCC Deposit No. 2 Wild type Deleted nucleotides chr5:PTA-125814 sequence 63301999-63302005 3 Wild type Deleted nucleotideschr5: PTA-125815 sequence 63301999-63302005 15 Wild type Deletednucleotides chr5: PTA-125816 sequence 63301999-63302005 65 Wild typeDeleted nucleotides chr5: PTA-125813 sequence 63301999-63302005Each of the animals in Table 16 presented a healthy phenotype withdeleted nucleotides 63301999-63302005 (in exon 2) from chromosome 5.

Additional cell lines were grown from collagenase treated ear notchsamples from unedited animals from PIC™ lines 19, 27, and 62, weredeposited with the ATCC. A representative sample of PIC™ Line 19 wasdeposited with the ATCC® on Apr. 3, 2019 and assigned ATTC® PatentDeposit Number PTA-125811. A representative sample of PIC™ Line 27 wasdeposited with the ATCC on Apr. 3, 2019 and assigned ATTC® PatentDeposit Number PTA-125907. A representative sample of PIC™ Line 62 wasdeposited with the ATCC on Apr. 3, 2019 and assigned ATTC® PatentDeposit Number PTA-125812. Each deposit was made according to theBudapest Treaty. Using conventional cloning methods, animals aregenerated from the cell lines deposited as PTA-125811, PTA-125812, andPTA-125907 and edited using the methods of Example 6 in order togenerate edited lines.

Single nucleotide polymorphisms (SNPs) in the vicinity of the CD163 genewere analyzed for each of the deposited PIC™ lines (2, 3, 15, 19, 27,62, and 65) and SNP profiles of each of the lines that are capable ofdistinguishing each line are selected. The starting dataset for definingthe line signatures was a collection of 330 whole genome sequencedanimals from the 7 deposited PIC™ lines. A 6 Mb region of Chromosome 5centered on the CD163 gene was extracted for the signatures, andvariation within and between the lines was examined for each nucleotidein this region in order to identify a relatively small number of SNPsthat together formed a signature for the line.

To be a candidate for inclusion in the signature of a given line, thefollowing criteria were imposed on each chromosomal position: sequencecoverage had to exist for 90% of animals in the line; the above had tobe true for at least 5 of the 7 lines; and all animals with data in thetarget line must have had the same homozygous genotype. For each of theother lines, a genotype frequency for this genotype was calculatedacross all the sequenced animals for that line. A cutoff was imposed ofat least 30% of the difference between the highest and lowest per-linegenotype frequency of the other 6 lines (i.e., there must be a spread ofgenotype frequencies within the 6 lines).

A combination of metrics for discriminating power and even distributionwere used to select a subset of genotypes that could define each line.Table 8 to Table 14 provide the positions on chromosome 5 for the SNPsfor which the homozygous allele was fixed in each porcine line. Thesesets of homozygous alleles distinguish each porcine line from the otherlines. The genotype listed indicates which allele is homozygous at eachposition, as indicated in Table 8 to Table 14.

Example 8

This example compares differing levels of PRRSv resistance of immunecells isolated from wild type and gene edited pigs.

CD163 surface expression analysis was conducted on Monocyte DerivedMacrophages (MoMØs) recovered from edited and wild type animals, eachwith and without edits to the CD163 gene according to the methodsdescribed in the previous examples. The edits tested are presented inTable 17. Four edits comprise deletions of various sizes as shown, andthe fifth comprises a 2 base pair insertion; all edits in Table 15 arein exon 2. All deletions or insertions result in a truncated CD163polypeptide. CD163 expression was assessed by immunofluorescencelabelling and FACS analysis using clone 2A10/11, a mouse anti-pig CD163monoclonal antibody. CD163 edited cells, MoMØs, lacked a functionalepitope on the surface of the cell as evident from cell surfaceexpression analysis.

TABLE 17 CD163 gene edits tested for protein expression Region Chr5Location of guideRNAs 63301997- 63301997- 63301997- 63301997- 63301997-binding site 02016 02016 02016 02016 02016 SEQ ID NO 14 15 16 17 18Guide RNA 42 42 42 42 42 spacer (DNA) SEQ ID NO Guide RNA 288 288 288288 288 spacer (RNA) SEQ ID NO Translation of 339 340 341 342 343Deletion Base deletion or insertion chr5: 6330201 chr5: 6330200 chr5:6330201 chr5: 6330200 chr5: 6330201 coordinates 2-63302013 9-633020412-63302013 5-63302020 1-63302020 Deleted or TC (2 bp SEQ ID TC (2 bp SEQID SEQ ID inserted bases deletion) NO: 344; insertion) NO: 345; NO: 346;(#) (33 bp (16 bp (10 bp deletion) deletion) deletion)

To test the two base pair deletion edit to CD163 in homozygous editedcells for PRRSv viral infection, MoMØs were infected with PRRSv type 1and type 2. Twenty-four (24) hours post infectivity, in a microscopefield of view, cells were counted to determine the number of cells whichcontain replicating PRRSv. CD163 homozygous edited MoMØcells were foundto be not permissive to both PRRSv types 1 and 2.

Example 9

This example demonstrates the use of two guides for removal of exon 7from S. scrofa CD163 in porcine fibroblasts.

In order to remove CD163 exon 7 DNA sequences which encode SRCR5 of themature CD163 polypeptide, DNA sequences located in the intronic regions450 bp upstream and 59 bp downstream of CD163 exon 7 were examined forStreptococcus pyogenes (NGG) and Streptococcus thermophilus (NGGNG) Cas9protein and guideRNA recognition sites. 48 sites were identified withinthe 450 bp of intron 6 and 10 sites were identified within a 59 bpregion of intron 7. The ability of these 58 sites to bind to Cas9 andguideRNAs to direct gene editing was first tested using single guides inporcine fetal fibroblasts. A subset of these single guideRNA-Cas9proteins—the guideRNAs which generated a high frequency of edits acrossthe spacer recognition site—were further tested as guide pairs for theirability to remove CD163 exon 7. Guides were introduced to porcine fetalfibroblasts by nucleofection as described in Example 2: each guide wasprepared as an RNP and then the two sets of complexes were combined in atotal volume of 2.23 μl prior to transfection. Editing frequency forguide pairs was determined as described in Example 3. The frequencies ofNHEJ-mediated repairs whereby the endonuclease cut sites are broughttogether resulting in the deletion of CD163 exon 7 are shown in Table18.

TABLE 18 End-to-end repair frequencies using paired guideRNAs for CD163exon 7 deletion in porcine fetal fibroblasts Desired SEQ SEQ repair IDNO: Cut site ID NO: Cut site Deletion outcome (5′) (5′) (3′) (3′) size(bp) (%) 229 63322816 256 63323377 561 23.3 230 63322814 256 63323377562 24.7 231 63322826 256 63323377 551 15.3 237 63322861 256 63323377516 20.7 241 63322891 256 63323377 486 20.0 229 63322816 258 63323378562 5.3 230 63322814 258 63323378 563 7.0 231 63322826 258 63323378 5524.3 237 63322861 258 63323378 517 15.7 241 63322891 258 63323378 487 5.7229 63322816 261 63323373 558 15.0 230 63322814 261 63323373 559 18.0231 63322826 261 63323373 548 0.0 237 63322861 261 63323373 513 12.0 24163322891 261 63323373 483 14.0 219 63322697 256 63323377 680 28.0 22163322709 256 63323377 668 33.7 224 63322747 256 63323377 630 27.0 22763322786 256 63323377 591 22.7 219 63322697 258 63323378 681 7.7 22163322709 258 63323378 669 13.0 224 63322747 258 63323378 631 7.3 22763322786 258 63323378 592 6.3 219 63322697 261 63323373 677 14.5 22163322709 261 63323373 665 20.7 224 63322747 261 63323373 627 14.7 22763322786 261 63323373 588 11.3 249 63322963 256 63323377 414 38.0 25063322973 256 63323377 404 36.7 249 63322963 258 63323378 415 20.3 25063322973 258 63323378 405 14.7 249 63322963 261 63323373 411 17.7 25063322973 261 63323373 401 11.7

The excision guides had a wide range of editing frequencies for thedesired edit.

Example 10

This example illustrates the excision of Exon 7 in porcine blastocystsusing dual guide RNAs.

A subset of guides screened in porcine fetal fibroblasts wereadditionally tested for their ability to remove CD163 exon 7 in porcineblastocysts. The subset of guides to be tested in porcine embryos waschosen based on a combination of their efficacy in generating exon 7deletions in porcine fibroblasts and low number of off-target edits foreach guide in the pair (see Detailed Description). Edited porcineembryos were generated as described above. Briefly, oocytes recoveredfrom slaughterhouse ovaries were in vitro fertilized as described inExample 6. The sgRNP solution was injected into the cytoplasm ofpresumptive zygotes at 4-5 hours post-fertilization by using a singlepulse from a FEMTOJET® 4i microinjector (Eppendorf; Hamburg, Germany)with settings at pi=200 hPa, ti=0.25 s, pc=15 hPa. Glass capillarypipettes (Sutter Instrument, Navato, Calif., USA) with an outer diameterof 1.2 mm and an inner diameter of 0.94 mm were pulled to a very finepoint of <0.5 Microinjection was performed in TL-Hepes (ABT360, LLC)supplemented with 3 mg/ml BSA (Proliant) on the heated stage of aninverted microscope equipped with Narishige (Narishige InternationalUSA, Amityville, N.Y.) micromanipulators. Following injections,presumptive zygotes were cultured for 7 days in PZM5 (Cosmo Bio, Co LTD,Tokyo, Japan) in an incubator environment of 5% CO₂, 5% O₂, 90% N₂.Editing frequency of blastocysts was determined as described in Example3. The frequencies of end-to-end NHEJ repairs resulting in the deletionof CD163 exon 7 are shown in Table 19.

TABLE 19 End-to-end repair frequencies using paired guideRNAs for inCD163 exon 7 deletion in porcine embryos Desired SEQ SEQ repair ID NO:Cut site ID NO: Cut site Deletion outcome (5′) (5′) (3′) (3′) size (bp)(%) 229 63322816 256 63323377 561 38.0 230 63322814 256 63323377 56221.0 231 63322826 256 63323377 551 24.0 241 63322891 256 63323377 48629.0 229 63322816 258 63323378 562 7.0 231 63322826 258 63323378 55212.0 241 63322891 258 63323378 487 20.0 219 63322697 256 63323377 68035.0 221 63322709 256 63323377 668 36.0 224 63322747 256 63323377 63024.0 227 63322786 256 63323377 591 0.0 227 63322786 258 63323378 592 0.0221 63322709 261 63323373 665 15.0 249 63322963 256 63323377 414 44.0250 63322973 256 63323377 404 14.0 249 63322963 258 63323378 415 7.0 24963322963 261 63323373 411 53.0

This example demonstrates that a number of guide pairs can be used todelete CD163 exon 7, but the efficiency can vary greatly between guidepairs and cell types.

Example 11

This example demonstrates the use of two guides to introduce a prematurestop codon into exon 7 of S. scrofa CD163.

Guides within exon 7 of CD163 were screened by bioinformatic methods fortheir ability to generate an in-frame stop codon when the cuts generatedby those guides are ligated together during NHEJ. Bioinformaticpredictions were tested in porcine fetal fibroblasts as described inExample 2: guides were complexed separately before being combined in atotal volume of 2.23 μl prior to transfection. Editing frequency wasdetermined as described in Example 3. The frequencies of end-to-end NHEJrepairs resulting in the introduction of a premature stop codon in exon7 of CD163 are shown in Table 20.

TABLE 20 End-to-end repair frequencies for introduction of a stop codonin CD163 exon 7 deletion in porcine fetal fibroblasts Desired Deletionrepair SEQ ID Cut site SEQ ID Cut site size outcome NO: (5′) (5′) NO:(3′) (3′) (bp) (%) 351 63323023 365 63323103 80 50.5 351 63323023 38763323235 212 1.0 348 63323027 390 63323236 209 53.5 348 63323027 38863323236 209 50.0 348 63323027 395 63323284 257 31.0 352 63323035 36563323103 68 29.0 352 63323035 387 63323235 200 12.5 352 63323035 39963323283 248 21.0 353 63323038 365 63323103 65 21.0 353 63323038 38763323235 197 6.0 353 63323038 399 63323283 245 11.0 354 63323039 39063323236 197 55.5 354 63323039 388 63323236 197 18.0 354 63323039 39563323284 245 17.0 358 63323056 361 63323077 21 0.0 358 63323056 36263323087 31 4.0 358 63323056 368 63323124 68 37.0 358 63323056 38463323203 147 34.0 358 63323056 394 63323272 216 56.0 358 63323056 39963323283 227 34.0 359 63323057 390 63323236 179 13.0 359 63323057 38863323236 179 3.0 359 63323057 395 63323284 227 4.0 360 63323058 36863323124 66 7.5 360 63323058 384 63323203 145 6.0 360 63323058 38963323237 179 0.0 360 63323058 394 63323272 214 16.5 360 63323058 39763323285 227 0.0 361 63323077 365 63323103 26 42.0 361 63323077 38763323235 158 0.0 362 63323087 390 63323236 149 45.0 362 63323087 38863323236 149 17.5 362 63323087 395 63323284 197 22.5 364 63323089 36563323103 14 0.0 364 63323089 387 63323235 146 14.5 364 63323089 39963323283 194 35.5 365 63323103 368 63323124 21 0.0 365 63323103 38463323203 100 17.0 365 63323103 389 63323237 134 0.0 365 63323103 39463323272 169 32.5 365 63323103 397 63323285 182 1.0 366 63323118 36863323124 6 0.0 366 63323118 384 63323203 85 29.0 366 63323118 38963323237 119 14.0 366 63323118 394 63323272 154 48.5 366 63323118 39763323285 167 10.0 354 63323039 211 63323163 123 29.0

This example illustrates that guide pairs designed to introduce a stopcodon have a wide variety of editing efficiencies in porcinefibroblasts.

Example 12

This example illustrates the editing efficiency of guides introducingstop codons in porcine blastocysts.

A subset of guides screened in porcine fetal fibroblasts wereadditionally tested for their ability to introduce a premature stopcodon in exon 7 of CD163 in porcine embryos. The subset of guides to betested in porcine embryos was chosen based on a combination of theirefficacy in introducing a premature stop codon in exon 7 of CD163 inporcine fibroblasts and the lack of observed off-targets for each guidein the pair as described supra. Editing frequency was determined asdescribed in Example 3. The frequencies of end-to-end NHEJ repairsresulting in the introduction of a premature stop codon in exon 7 ofCD163 are shown in Table 21.

TABLE 21 End-to-end repair frequencies for introduction of a stop codonin CD163 exon 7 deletion in porcine blastocysts SEQ ID Cut site SEQ IDCut site Deletion Desired repair NO: (5′) (5′) NO: (3′) (3′) size (bp)outcome (%) 351 63323023 365 63323103 80 39.6 348 63323027 390 63323236209 31 348 63323027 388 63323236 209 35.4 354 63323039 390 63323236 19730.4 358 63323056 394 63323272 216 29.2 362 63323087 390 63323236 149 31366 63323118 394 63323272 154 33 354 63323039 211 63323163 123 27.4

This example demonstrates that guide pairs that can be used to introducea premature stop codon into exon 7 of CD163 have a wide variety ofediting efficiencies.

Example 13

This example illustrates variable repair outcomes for NHEJ repair.

A subset of guideRNAs designed to delete CD163 exon 7 were tested fortheir ability to delete the exon 7 coding and flanking regions inporcine blastocysts, as described in Example 9. Editing frequency ofblastocysts was determined as described in Example 3. In this example, asubset of the DNA sequences observed in porcine blastocysts in vivo asthe result of NHEJ-mediated repair are shown for five guideRNA pairs inTable 7. It is known that simple nucleotide deletions also occur, aswell as more complex NHEJ-mediated repair DNA sequences which containdeletions, insertions, rearrangements, inversions, and any combinationthereof. Without being limited by theory, these varied repair outcomesalso occur as a result of endonuclease cutting of DNA using single andpaired guides in porcine blastocysts. Table 7 also shows that thefrequencies of the observed DNA sequence associated with each repairoutcome vary between guideRNA pairs. In some, but not all, instances thefrequency of DNA sequence associated with the end-to-end joining of cutsites of the paired guideRNA was the most highly represented repairoutcome. For some guideRNA pairs there was a single predominant DNArepair outcome, while for other guideRNA pairs there were DNA repairoutcomes that occurred with equal frequency. Most of the DNA repairoutcomes shown for these five guideRNA pairs resulted in the deletion ofCD163 exon 7 DNA sequences corresponding to the SRCR 5 domain.Therefore, the decision for which guideRNA pair to use for generation ofedited pigs depends largely on the tolerance for multiple alleles in apopulation.

This example demonstrates that the NHEJ-mediated repair outcomes thatresult when two intronic guideRNAs are used to delete genomic DNA varybetween guideRNA pairs, not only in the manner in which DNA breaks areresolved but in the frequency of these resolutions. It is advantageousto screen guideRNA pairs in fibroblasts or embryos to observe DNA repairoutcomes of single or dual guideRNAs for the generation of editedanimals.

Example 14

This example illustrates a real time PCR assay for identifying thepresence of the spacer sequence set forth in SEQ ID NO: 249 and/or thedesired Exon 7 excision edit in cells.

Two sets of primers and two probes were designed for this assay. One setof primers were designed to flank the spacer sequence set forth in SEQID NO: 249 in the unedited genome. The sequences of these primers areset forth in SEQ ID NOs: 556 and 557. A probe of sequence SEQ ID NO: 558and labeled with the HEX fluorescent moiety was designed to anneal tothe unedited genome between the PCR primers. The other set of primers,having sequences set forth in SEQ ID NOs: 562 and 563, were designed toflank the desired edit sequence. A probe, having a sequence set forth inSEQ ID NO: 564 and labeled with the FAM fluorescent moiety, was designedto anneal to nucleotides spanning the joining region of the edit. Realtime PCR was performed using both primer sets and with genomic DNAextracted from tail and/or ear samples of pigs that had known allelicstatus (wild type, homozygous, or heterozygous). 5 μl of 2× PRIMETIME®Master Mix (Integrated DNA Technologies, Coralville, Iowa) was mixedwith 0.5 μl of each primer (10 μM), 0.5 μl of each probe (2.5 μM), and 2μl of genomic DNA. PCR was performed with 3 minutes initial denaturingat 95° C., then 35 cycles of: 95° C. for 15 seconds, 64° C. for 30seconds, and 72° C. for 30 seconds. A final elongation was performed at72° C. for 2 minutes, and then the reaction was held in the cycler at 4°C. Fluorescence was measured and charted and, as expected, thehomozygotes were close to the y axis (representing the FAM moietywavelength), the heterozygotes grouped near the center of the chart, andthe wild type pigs grouped close to the X axis (representing the HEXmoiety wavelength). The assay was therefore successful in detecting theedit based on the spacer sequence set forth in SEQ ID NO: 249.

Example 15

This example illustrates a real time PCR assay for identifying thepresence of the spacer sequence set forth in SEQ ID NO: 256 and/or thedesired Exon 7 excision edit in cells.

Two sets of primers and two probes were designed for this assay. One setof primers flanked the spacer sequence set forth in SEQ ID NO: 256. Thesequence of these primers is set forth in SEQ ID NOs: 559 and 560. Aprobe, having a sequence set forth in SEQ ID NO: 561 and labeled withthe HEX fluorescent moiety, was designed to anneal to the uneditedversion of the spacer sequence. The other set of primers and probe aredesigned to target the desired edit and are described in Example 14 (SEQID NOs: 562-564.) Real time PCR was performed as described in Example14, but with the instant primers and probes. Fluorescence was chartedand, as expected, the homozygotes were close to the y axis (representingthe FAM moiety), the heterozygotes grouped near the center of the chart,and the wild type pigs grouped close to the X axis (representing the HEXmoiety). The assay was therefore successful in detecting the edit basedon the spacer sequence set forth in SEQ ID NO: 256.

Example 16

This example illustrates a comparison of CD163 CRISPR-CAS gRNA activityin cells between previously published guide pairs and guide pairs of thepresent teachings.

Each pair of guides was tested in porcine fibroblasts as described inExample 2. Each pair was further tested in porcine blastocysts asdescribed in Example 10. The results are shown below in Table 22.

TABLE 22 Comparative Activity in Porcine Cells guide pair % desired %desired SEQ ID repair repair NOs: Source fibroblasts blastocysts 272 and273 Burkard 2017 6 20 249 and 256 Present Disclosure 38 44 354 and 211Whitworth 2014 17 n.d. 362 and 390 Present Disclosure 45 31

These data illustrate that the guides of the present disclosure provideat least a two-fold improvement in the percentage of cells that have thedesired edit relative to the guides that were previously disclosed inthe literature. Therefore, the guides of the present disclosure are moreefficient than the guides previously disclosed in the literature.

Example 17

This example illustrates CD163 edited pigs challenged with PRRSv Type I.

Pigs from PIC™ Line 2 were edited with guides as set forth in SEQ IDNOs: 249 and 256 as described in Example 6. Edits were confirmed asdescribed in Example 5. Edited pigs were then crossbred to create pigsthat were homozygous for the edit. These homozygous edited pigs wereinoculated with 3 ml of PRSSv Type I (SD13-15) having 10⁴ to 10⁵ virions(4-5 log TCID₅₀). 1.5 ml was administered intramuscularly with a 21gauge needle. The remaining 1.5 ml was administered intranasally. Serumsamples were obtained on Day 0 (prior to inoculation on that day), Day3, Day 5, Day 10, Day 14, and Day 21. Realtime PCR to determine thepresence of virus in the serum samples using TETRACORE® EZ-PRRSV MPX 4.0Master Mix and Enzyme with ROX (TETRACORE®, Rockville, Md.) according tomanufacturer directions. The real time PCR EU adjusted counts are shownin Table 23. The counts have been inverted using standard methods knownin the art to make the data more intuitive—higher numbers indicate morevirus detected.

TABLE 23 Realtime PCR EU Adjusted Counts for Type I PRRSv ChallengeAnimal CD163 Day 0 Day 3 Day 5 Day 10 Day 14 Day 21 2 WT 0 19.8 22.6 2019.6 18.3 4 WT 0 17 17.7 18.6 16.8 17.5 5 WT 0 18.9 21.5 17.3 14.2 13.66 WT 0 15.8 19.7 15.6 14.5 13.4 17 WT 0 17.1 17.8 18.5 18 15.8 18 WT 015.8 17.9 17 16.8 15 20 WT 0 17.2 20.3 18.8 17 12.8 3 Edit 0 0 0 0 0 0 7Edit 0 0 0 0 0 0 14 Edit 0 0 0 0 0 0 19 Edit 0 0 0 0 0 0

No PRRSv was detected in the serum of the edited animals throughout theexperiment.

The serum samples were also subjected to ELISA using the IDEXX PRRS X3antibody test kit; the test was performed by an accredited VeterinaryDiagnostic Laboratory. The results are shown as a Sample:Positive ratio.Ratios greater than or equal to 0.40 are considered positive. The ratiosfor each sample are shown in Table 24.

TABLE 24 ELISA S/P Results for Type I PRRSv Challenge Animal CD163 Day 0Day 3 Day 5 Day 10 Day 14 Day 21 2 WT 0.0 0.0 0.0 0.5 0.6 1.4 4 WT 0.00.0 0.0 0.1 0.2 0.8 5 WT 0.0 0.0 0.0 0.9 1.3 1.8 6 WT 0.0 0.0 0.0 1.31.3 1.9 17 WT 0.0 0.0 0.0 0.2 0.2 1.1 18 WT 0.0 0.0 0.0 0.3 0.3 1.4 20WT 0.0 0.0 0.0 0.3 0.4 1.2 3 Edit 0.0 0.0 0.0 0.0 0.0 0.0 7 Edit 0.0 0.00.0 0.0 0.0 0.0 14 Edit 0.0 0.0 0.0 0.0 0.0 0.0 19 Edit 0.0 0.0 0.0 0.00.0 0.0

The edited pigs do not have any positive ratios. In contrast, by day 21,all of the wild type pigs have positive ratios. This further illustratesthat there is no PRRSv circulating in the edited pigs' serum.

This example illustrates that pigs edited with guides of SEQ ID NOs: 249and 256 are resistant to PRRSv Type I virus infection.

Example 18

This example illustrates CD163 edited pigs challenged with PRRSv TypeII.

Pigs from PIC™ Lines 2 and 3 were edited with guides as set forth in SEQID NOs: 249 and 256 as described in Example 6. Edits were confirmed asdescribed in Example 5. Edited pigs were then crossbred to create pigsthat were homozygous for the edit. These homozygous edited pigs werethen inoculated with 3 ml of PRRSv Type II (NVSL 97-7895) having 10⁴ to10⁵ virions (4-5 log TCID₅₀). 1.5 ml was administered intramuscularlywith a 21 gauge needle. The remaining 1.5 ml was administeredintranasally. Serum samples were obtained on Day 0 (prior to inoculationon that day), Day 3, Day 5, Day 10, Day 14, and Day 21. Realtime PCR todetermine the presence of virus in the serum samples using TETRACORE®EZ-PRRSV MPX 4.0 Master Mix and Enzyme with ROX according tomanufacturer directions. The NA adjusted counts for real time PCR areshown in

Table 25. The numbers were inverted to make them more intuitive—thehigher the count, the more virus is present.

TABLE 25 Realtime PCR NA Adjusted Counts for Type II PRRSv ChallengeAnimal CD163 Day 0 Day 3 Day 5 Day 10 Day 14 Day 21 21 WT 0.0 18.4 22.021.9 18.0 16.1 23 WT 0.0 16.4 22.0 21.8 20.0 19.7 25 WT N/A 17.2 21.221.9 19.2 19.8 30 WT 0.0 16.2 17.8 21.4 21.1 17.6 33 WT 0.0 18.5 N/A21.8 18.5 15.3 35 WT N/A 17.0 22.9 21.8 20.5 20.9 36 WT 0.0 16.7 21.421.9 19.9 17.4 38 WT 0.0 16.0 18.1 20.2 20.9 9.4 24 Edit N/A 1.9 0.0 0.00.0 0.0 37 Edit 0.0 0.0 0.0 0.0 0.0 0.0

The edited pigs have very little to no virus counts relative to the wildtype pigs.

The serum samples were also subjected to ELISA using the IDEXX PRRS X3antibody test kit; the test was performed by an accredited VeterinaryDiagnostic Laboratory. The results are shown as a Sample:Positive ratio.Ratios greater than or equal to 0.40 are considered positive. The ratiosfor each sample are shown in Table 26.

TABLE 26 ELISA S/P Ratios for Type II PRRSv Challenge Animal Edit Day 0Day 3 Day 5 Day 10 Day 14 Day 21 21 WT 0.0 0.0 0.0 1.7 2.0 1.9 23 WT 0.00.0 0.0 1.2 1.7 1.9 25 WT N/A 0.0 0.0 1.2 1.6 1.7 30 WT 0.0 0.0 0.0 0.40.6 0.6 33 WT 0.0 0.0 N/A 0.7 0.6 0.7 35 WT N/A 0.0 0.0 1.3 1.3 1.4 36WT 0.0 0.0 0.0 1.2 1.5 1.8 38 WT 0.0 0.0 0.0 0.5 0.9 1.0 24 Edit N/A 0.20.1 0.2 0.1 0.1 37 Edit 0.0 0.0 0.0 0.0 0.0 0.0

The edited pigs do not have any positive ratios; in contrast, by day 10,all of the wild type pigs have positive ratios. Taken together, thesedata illustrate that there is no virus circulating in the edited pigs'blood.

This example illustrates that the pigs with a CD163 gene edited with SEQID NOs: 249 and 256 are resistant to PRRSv Type II infection.

The contents of each of the foregoing references and applications areincorporated herein by reference in their entireties. Having describedthe present disclosure in detail, it will be apparent that modificationsand variations are possible without departing from the scope of theteachings defined in the appended claims.

What is claimed is:
 1. A method of making a progeny Sus scrofa having agenome containing a non-wild-type, edited CD163 gene comprising sequenceSEQ ID NO: 453, the method comprising the steps of: obtaining a geneticsample of a first Sus scrofa; determining the presence or absence of SEQID NO: 453 in the genome of the first Sus scrofa; selecting the firstSus scrofa for breeding if SEQ ID NO: 453 is present in the genome ofthe first Sus scrofa; and breeding the first Sus scrofa with a secondSus scrofa of the opposite sex to generate the progeny Sus scrofa havinga genome containing a non-wild-type, edited CD163 gene comprisingsequence SEQ ID NO:
 453. 2. The method of claim 1, wherein thedetermining comprises annealing of a probe to SEQ ID NO:
 453. 3. Themethod of claim 1, wherein the determining comprises at least one realtime PCR (rtPCR) reaction comprising a probe that anneals to SEQ ID NO:453.
 4. The method of claim 1, wherein the determining comprises tworeal time PCR (rtPCR) reactions, each reaction using a differentlylabelled probe; wherein a first probe anneals to SEQ ID NO: 453 and asecond probe anneals to SEQ ID NO: 249, wherein: a) detecting signalfrom the label of the first probe and not the label of the second probeindicates a homozygous edit; b) detecting signals from the labels of thefirst probe and the second probe indicates a heterozygous edit; and c)detecting signal from the label of the second probe and not the label ofthe first probe indicates a homozygous wild type genome.
 5. The methodof claim 4, wherein the rtPCR reaction with the second probe comprisesprimers having sequences consisting of SEQ ID NO: 556 and SEQ ID NO:557.
 6. The method of claim 4, wherein the rtPCR reaction with the firstprobe comprises primers having nucleotide sequences consisting of SEQ IDNO: 562 and SEQ ID NO:
 563. 7. The method of claim 4, wherein the secondprobe has a nucleotide sequence consisting of SEQ ID NO:
 558. 8. Themethod of claim 4, wherein the first probe has a nucleotide sequenceconsisting of SEQ ID NO:
 564. 9. The method of claim 4, wherein the twortPCR reactions comprise primers having nucleotide sequences consistingof SEQ ID NO: 562, SEQ ID NO: 563, SEQ ID NO: 556, and SEQ ID NO: 557.10. The method of claim 1, wherein the Sus scrofa was edited as an MIIoocyte or zygote.
 11. The method of claim 1, wherein the determiningcomprises: a first real time PCR (rtPCR) reaction comprising: primershaving nucleotide sequences consisting of SEQ ID NO: 562 and SEQ ID NO:563; and a first labelled probe having a sequence consisting of SEQ IDNO: 564; and a second rtPCR reaction comprising: primers havingnucleotide sequences consisting of SEQ ID NO: 556 and SEQ ID NO: 557;and a second labelled probe having a sequence consisting of SEQ ID NO:558 wherein the label is different than that of the first probe.
 12. ASus scrofa progeny obtained by the method of claim
 1. 13. The method ofclaim 1, wherein: the determining comprises two real time PCR (rtPCR)reactions, each reaction using a differently labelled probe; wherein afirst probe anneals to SEQ ID NO: 453 and a second probe anneals to SEQID NO: 256, wherein: a) detecting signal from the label of the firstprobe and not the label of the second probe indicates a homozygous edit;b) detecting signals from the labels of the first probe and the secondprobe indicates a heterozygous edit; and c) detecting signal from thelabel of the second probe and not the label of the first probe indicatesa homozygous wild type genome.
 14. The method of claim 13, wherein thertPCR reaction with the second probe comprises primers having nucleotidesequences consisting of SEQ ID NO: 559 and SEQ ID NO:
 560. 15. Themethod of claim 13, wherein the rtPCR reaction with the first probecomprises primers having nucleotide sequences consisting of SEQ ID NO:562 and SEQ ID NO:
 563. 16. The method of claim 13, wherein the secondprobe has a nucleotide sequence consisting of SEQ ID NO:
 561. 17. Themethod of claim 13, wherein the first probe has a nucleotide sequenceconsisting of SEQ ID NO:
 564. 18. The method of claim 13, wherein thetwo rtPCR reactions comprise primers having nucleotide sequencesconsisting of SEQ ID NO: 562, SEQ ID NO: 563, SEQ ID NO: 559, and SEQ IDNO:
 560. 19. The method of claim 1, wherein the determining comprises: afirst real time PCR (rtPCR) reaction comprising: primers havingnucleotide sequences consisting of SEQ ID NO: 562 and SEQ ID NO: 563;and a first labelled probe having a sequence consisting of SEQ ID NO:564; and a second rtPCR reaction comprising: primers having nucleotidesequences consisting of SEQ ID NO: 559 and SEQ ID NO: 560; and a firstlabelled probe having a sequence consisting of SEQ ID NO: 561 whereinthe label is different than that of the first oligonucleotide probe. 20.An isolated nucleic acid selected from the group consisting of SEQ IDNO: 556, SEQ ID NO: 557, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 560,SEQ ID NO: 561, SEQ ID NO: 562, SEQ ID NO: 563, SEQ ID NO: 564.